Binding proteins, including antibodies, antibody derivatives and antibody fragments, that specifically bind cd154 and uses thereof

ABSTRACT

This invention provides binding proteins, including antibodies, antibody derivatives and antibody fragments, that specifically bind a CD154 (CD40L) protein. This invention also provides a chimeric, humanized or fully human antibody, antibody derivative or antibody fragment that specifically binds to an epitope to which a humanized Fab fragment comprising a variable heavy chain sequence according to SEQ ID NO: 1 and comprising a variable light chain sequence according to SEQ ID NO: 2 specifically binds. CD154 binding proteins of this invention may elicit reduced effector function relative to a second anti-CD154 antibody. CD154 binding proteins of this invention are useful in diagnostic and therapeutic methods, such as in the treatment and prevention of diseases including those that involve undesirable immune responses that are mediated by CD154-CD40 interactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/084,088, filedMar. 29, 2016, which is a continuation of U.S. Ser. No. 14/309,118,filed Jun. 19, 2014, now U.S. Pat. No. 9,321,840, which is a divisionalof U.S. Ser. No. 13/656,922, filed Oct. 22, 2012, now U.S. Pat. No.8,784,823, which is a continuation of U.S. Ser. No. 12/532,517, filedSep. 22, 2009, now U.S. Pat. No. 8,293,237, which is the U.S. nationalstage application of International Application PCT/US2008/003735, filedMar. 21, 2008, which claims priority from U.S. Provisional PatentApplication No. 60/920,495, filed Mar. 27, 2007, U.S. Provisional PatentApplication No. 60/919,938, filed Mar. 22, 2007 and U.S. ProvisionalPatent Application No. 60/919,816, filed Mar. 22, 2007. The contents ofPCT/US2008/003735, U.S. 60/920,495, U.S. 60/919,938 and U.S. 60/919,816are hereby incorporated by reference in their entirety.

The Sequence Listing for this application is labeled “Seq-List.TXT”which was created on Apr. 7, 2019 and is 81 KB. The entire contents ofthe sequence listing are incorporated herein by reference in theirentirety.

TECHNICAL FIELD OF THE INVENTION

This invention provides binding proteins, including antibodies, antibodyderivatives and antibody fragments, that specifically bind a CD154(CD40L) protein. This invention also provides a chimeric, humanized orfully human antibody, antibody derivative or antibody fragment thatspecifically binds to an epitope to which a humanized Fab fragmentcomprising a variable heavy chain sequence according to SEQ ID NO: 1 andcomprising a variable light chain sequence according to SEQ ID NO: 2specifically binds. CD154 binding proteins of this invention may elicitreduced effector function relative to a second anti-CD154 antibody.CD154 binding proteins of this invention are useful in diagnostic andtherapeutic methods, such as in the treatment and prevention of diseasesincluding those that involve undesirable immune responses that aremediated by CD154-CD40 interactions.

BACKGROUND OF THE INVENTION

The generation of humoral and cell-mediated immunity is orchestrated bythe interaction of activated helper T cells with antigen-presentingcells (“APCs”) and effector T cells. Activation of the helper T cells isnot only dependent on the interaction of the antigen-specific T-cellreceptor (“TCR”) with its cognate peptide-MHC ligand, but also requiresthe coordinate binding and activation by a number of cell adhesion andcostimulatory molecules. See, e.g., Salazar-Fontana, L. I., and B. E.Bierer (2001) Curr. Opin. Hemat. 8:5.

One critical costimulatory molecule is CD154, a Type II transmembraneprotein that is expressed on the surface of CD4⁺ T cells in anactivation-dependent, temporally-restricted manner. CD154 is alsoexpressed, following activation, on a subset of CD8⁺ T cells, basophils,mast cells, eosinophils, natural killer cells, B cells, macrophages,dendritic cells and platelets. The CD154 counter-receptor, CD40, is aType I membrane protein that is constitutively and widely expressed onthe surface of many cell types, including APCs. See, e.g., Foy, T. M. etal. (1996) Ann. Rev. Immunol. 14:591.

Signaling through CD40 by CD154 initiates a cascade of events thatresults in the activation of the CD40 receptor-bearing cells and optimalCD4⁺ T cell priming. More specifically, the binding of CD154 to CD40promotes the differentiation of B cells into antibody secreting cellsand memory B cells. See, e.g., Burkly, L. C. (2001) in Adv. Exp. Med.Bio., Vol. 489. D. M. Monroe et al. eds. Kluwer Academic/PlenumPublishers, p. 135 (hereafter “Burkly, supra”). Additionally, theCD154-CD40 interaction promotes cell-mediated immunity through theactivation of macrophages and dendritic cells and the generation ofnatural killer cells and cytotoxic T lymphocytes. See, e.g., Burkly,ibid.

The pivotal role of CD154 in regulating the function of both the humoraland cell-mediated immune response has provoked great interest in the useof inhibitors of this pathway for therapeutic immunomodulation. As such,anti-CD154 antibodies have been shown to be beneficial in a wide varietyof models of immune response to other therapeutic proteins or genetherapy, allergens, autoimmunity and transplantation. See, e.g., U.S.Pat. No. 5,474,771; Burkly, supra.

The CD40-CD154 interaction has been shown to be important in severalexperimentally induced autoimmune diseases where it has been shown thatdisease induction can be blocked with CD154 antagonists at the time ofantigen administration (Burkly, supra). The blockade of disease usinganti-CD154 antagonists has also been seen in animal models ofspontaneous autoimmune disease. See, e.g., Burkly, supra.

There is currently a need for improved anti-CD154 antibodies with higherbinding affinities and fewer unwanted side effects. Increased “effectorfunctions” such as direct cytotoxicity, complement-dependentcytotoxicity (“CDC”), antibody-dependent cytotoxicity (“ADCC”) andabnormal antibody production are unwanted side effects that may beassociated with therapeutic antibodies.

Several antibody effector functions are mediated at least in part by Fcreceptors (FcRs), which bind the Fc region of an antibody in theconstant domain of a typical immunoglobulin. There are a number of Fcreceptors which are specific for the different classes ofimmunoglobulins. The classes of immunoglobulins include IgG, IgE, IgA,IgM, and IgD. The classes of immunoglobulins are further divided intosubclasses: IgG is divided into four subclasses (IgG1, IgG2, IgG3, andIgG4) and IgA is divided into two subclasses (IgA1 and IgA2). There arethree known receptors for IgG: FcγRI (CD64), FcγRII (CD32), and FcγRIII(CD16). Each FcγR subclass is encoded by two or three genes, andalternative RNA splicing leads to multiple transcripts and a broaddiversity in FcγR isoforms.

Typically, immunoglobulins are Y-shaped molecules comprising twoidentical heavy chains and two identical light chains. Disulfide bondslink together the heavy and light chain pairs as well as the two heavychains. Each chain consists of one variable domain that varies insequence and is responsible for antigen binding; these domains are knownas the V_(H) and V_(L) domains for the heavy and light chains,respectively. In the light chain there is a single constant domain(C_(L)) and in the heavy chain there are three constant domains (C_(H1),C_(H2) and C_(H3)). Molecules containing all of the variable andconstant domains may be referred to as whole antibodies.

The residues in antibody variable domains are conventionally numberedaccording to a system devised by Kabat et al. This system is set forthin Kabat et al., 1987, Sequences of Proteins of Immunological Interest,US Department of Health and Human Services, NIH, USA (hereafter “Kabatet al., supra”). This numbering system is used in the presentspecification except where otherwise indicated. It should be noted thatthe Kabat residue designations do not always correspond directly withthe linear numbering of the amino acid residues. The actual linear aminoacid sequence may contain fewer or additional amino acids than in thestrict Kabat numbering corresponding to a shortening of, or insertioninto, a structural component, whether framework or complementaritydetermining region (CDR), of the basic variable domain structure. Thecorrect Kabat numbering of residues may be determined for a givenantibody by alignment of residues of homology in the sequence of theantibody with a “standard” Kabat numbered sequence.

There are three regions within the variable domains that arehypervariable in sequence set within four more highly conservedframework regions. These hypervariable CDRs are primarily responsiblefor antigen recognition. The CDRs of the heavy chain variable domain arelocated at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues95-102 (CDR-H3) according to the Kabat numbering system. However,according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol., 1987,196:901-917), the loop equivalent to CDR-H1 extends from residue 26 toresidue 32. Thus “CDR-H1”, as used herein, also includes a CDR locatedat residues 26 to 35, as described by a combination of the Kabatnumbering system and Chothia's topological loop definition. The CDRs ofthe light chain variable domain are located at residues 24-34 (CDR-L1),residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to theKabat numbering system.

Though naturally occurring antibodies, as whole antibodies or asfragments retaining specific binding properties, were originally derivedfrom a single species, engineered antibodies may be derived from morethan one species of animal, e.g., chimeric antibodies. To date, mouse(murine)/human chimeric and murine/non-human primate antibodies haveprincipally been generated, though other hybrid species combinations arepossible. Many different configurations of naturally occurring andengineered antibody polypeptides, and derivatives and fragments thereof,are now known. The feature common to all is that the polypeptide orpolypeptides retain antigen-binding specificity through one or moreepitope-binding domains. Aside from epitope binding, the functionalproperties of an antibody polypeptide may differ depending on what othersequences are present, e.g., Fc domains or other sequences that activateeffector functions and/or interact with other cellular pathways.

CD154 binding proteins that comprise epitope-binding domains (such asCDRs or variable domains) incorporated into a non-immunoglobulinscaffold or framework (see, for example, Binz et al. 2005 Nat Biotech23: 1257-1268; Hosse et al. 2006 Protein Science 15: 14-27) may exhibitreduced effector functions. It would be desirable to have new bindingproteins that specifically antagonize CD154 binding to CD40 and reduceor eliminate downstream functions of the CD154-CD40 complex. It wouldalso be desirable to have CD154 binding proteins, such as anti-CD154antibodies, with reduced effector functions compared to known anti-CD154antibodies.

SUMMARY OF THE INVENTION

The present invention provides a solution to these and other problems byproviding a binding protein, such as an isolated, recombinant orsynthetic antibody, or fragment or derivative thereof, that specificallybinds a CD154 protein, which may be a human CD154 protein. Anti-CD154antibodies of the invention, including fragments and derivativesthereof, may be monoclonal, polyclonal, murine, chimeric, primatized,humanized or fully human antibodies. Anti-CD154 antibodies of theinvention may be multimeric, heterodimeric, hemidimeric, monovalent,bivalent, tetravalent or bispecific and may include single chainantibodies and derivatives thereof.

In certain embodiments, CD154 binding proteins, e.g., anti-CD154antibodies, of the present invention have high selectivity for CD154and, in some embodiments, also have one or more reduced effectorfunctions compared, for example, to anti-CD154 antibody 5c8 (produced bythe hybridoma deposited under ATCC Accession No. HB 10916, as describedin U.S. Pat. No. 5,474,771, or humanized 5c8). Certain of the CD154binding proteins, e.g., anti-CD154 antibodies, of the invention aremonovalent for CD154 binding.

CD154 binding proteins and anti-CD154 antibodies (including antibodyfragments and derivatives) of the invention are useful for inhibitingbinding of CD154 to CD40 and do so with high specificity, e.g., with anIC50 in the range of 20 pM to 1.5 μM, inclusive. In certain embodiments,the CD154 binding proteins, e.g., anti-CD154 antibodies, of theinvention may have an IC50 in the range of 20 pm to 500 pm, 50 pm to 500pm or 100 pm to 500 pm. In certain embodiments, CD154 binding proteinsand anti-CD154 antibodies (including antibody fragments and derivatives)of the invention do not substantially agonize CD40 activity.

Certain embodiments of the present invention relate to CD154 bindingproteins, e.g., anti-CD154 antibodies, that exhibit a high affinity forhuman CD154. For example, in certain embodiments, a CD154 bindingprotein, e.g., anti-CD154 antibody, dissociates from human CD154 (humanCD40L) with a K_(D) in the range of 50 nM to 1 pM, inclusive, asdetermined by surface plasmon resonance (e.g. BIACORE® (an assay tomeasure molecular interactions)). For example, the K_(D) for human CD154may be 50 pM to 1 pM, 20 pM to 1 pM or even 10 pM to 1 pM. In someembodiments, the K_(D) for human CD154 is less than 20 pM. In otherembodiments, the K_(D) for human CD154 is less than 10 pM.

In certain embodiments, this invention provides a CD154 binding protein,e.g., anti-CD154 antibody, which when present at or above saturatingconcentrations for CD154 binding based on its binding affinity iscapable of blocking binding of antibody 5c8 to CD154 when added to CD154first, and is also capable of displacing antibody 5c8 bound to CD154when added after antibody 5c8 is added to CD154.

In certain embodiments, this invention provides a binding protein, e.g.,antibody, that specifically binds a CD154 protein and which comprises orconsists of one or more CDR heavy chain (H) sequence(s) selected fromCDR-H1 (SEQ ID NO: 3), CDR-H2 (SEQ ID NO: 4) and CDR-H3 (SEQ ID NO: 5).In further embodiments, the CD154 binding protein or antibody comprisesor consists of at least two CDRs selected from CDR-H1 (SEQ ID NO: 3),CDR-H2 (SEQ ID NO: 4) and CDR-H3 (SEQ ID NO: 5). In yet furtherembodiments the binding protein or antibody comprises or consists of allthree CDR H sequences, which are the CDR-H1 (SEQ ID NO: 3), the CDR-H2(SEQ ID NO: 4) and the CDR-H3 (SEQ ID NO: 5).

In certain embodiments, this invention provides a binding protein, e.g.,antibody, that specifically binds a CD154 protein and which comprises orconsists of one or more CDR light chain (L) sequence(s) selected fromCDR-L1 (SEQ ID NO: 6), CDR-L2 (SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8).In further embodiments, the CD154 binding protein or antibody comprisesor consists of at least two CDRs selected from CDR-L1 (SEQ ID NO: 6),CDR-L2 (SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8). In yet furtherembodiments, the CD154 binding protein or antibody comprises or consistsof all three CDR L sequences, which are CDR-L1 (SEQ ID NO: 6), CDR-L2(SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8).

In certain embodiments, this invention provides a binding protein, e.g.,antibody, that specifically binds a CD154 protein and that comprises orconsists of CDR-L1 (SEQ ID NO: 6), CDR-L2 (SEQ ID NO: 7) and CDR-L3 (SEQID NO: 8), and wherein the binding protein or antibody further comprisesor consists of CDR-H1 (SEQ ID NO: 3), CDR-H2 (SEQ ID NO: 4) and CDR-H3(SEQ ID NO: 5).

In certain embodiments, this invention provides a binding protein, e.g.,antibody, that specifically binds a CD154 protein, wherein the CD154binding protein or antibody comprises or consists of a V_(H) sequenceselected from SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11. In certain other embodiments, this invention provides a bindingprotein, e.g., antibody, that specifically binds a CD154 protein,wherein the CD154 binding protein or antibody comprises or consists of aheavy chain sequence selected from SEQ ID NO: 12 and SEQ ID NO: 13.

In certain embodiments, this invention provides a binding protein, e.g.,antibody, that specifically binds a CD154 protein, wherein the CD154binding protein or antibody comprises or consists of a V_(L) sequenceselected from SEQ ID NO: 2 and SEQ ID NO: 14. In certain embodiments,this invention provides a binding protein, e.g., antibody, thatspecifically binds a CD154 protein, wherein the CD154 binding protein orantibody comprises or consists of the light chain sequence of SEQ ID NO:15.

In certain embodiments, this invention provides a binding protein, e.g.antibody, that specifically binds a CD154 protein, wherein the CD154binding protein or antibody comprises or consists of a V_(L) sequenceselected from SEQ ID NO: 2 and SEQ ID NO: 14 and a V_(H) sequenceselected from SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11. In certain embodiments, this invention provides a binding protein,e.g., antibody, that specifically binds a CD154 protein, wherein theCD154 binding protein or antibody comprises or consists of a light chainsequence of SEQ ID NO: 15 and a heavy chain sequence selected from SEQID NO: 12 and SEQ ID NO: 13. In other embodiments, the CD154 bindingprotein or antibody comprises or consists of a light chain sequence ofSEQ ID NO: 15 and a heavy chain sequence of SEQ ID NO: 13.

In other embodiments, the present invention relates to a bindingprotein, e.g. an antibody, that specifically binds CD154, and comprisesor consists of one or more heavy chain (H) CDR sequence(s) selected fromCDR-H1 (SEQ ID NO: 42), CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQ ID NO:44). In further embodiments, the binding protein or antibody comprisesor consists of at least two CDRs selected from CDR-H1 (SEQ ID NO: 42),CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQ ID NO: 44). In yet furtherembodiments, the binding protein or antibody comprises or consists ofall three CDR H sequences, which are CDR-H1 (SEQ ID NO: 42), CDR-H2 (SEQID NO: 43) and CDR-H3 (SEQ ID NO: 44).

In certain embodiments, this invention provides a binding protein, e.g.an antibody, that specifically binds a CD154 protein, wherein thebinding protein or antibody comprises or consists of one or more CDRlight chain (L) sequence(s) selected from CDR-L1 (SEQ ID NO: 45), CDR-L2(SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47). In further embodiments, thebinding protein or antibody comprises or consists of at least two CDRsselected from CDR-L1 (SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3(SEQ ID NO: 47). In yet further embodiments, the binding protein orantibody comprises or consists of all three CDR L sequences, which areCDR-L1 (SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO:47).

In certain embodiments, a CD154 binding protein or anti-CD154 antibodyof the present invention comprises a complementary sequence comprisingor consisting of one or more light chain CDRs of CDR-L1, CDR-L2 andCDR-L3, above, or a complementary sequence comprising or consisting ofone or more heavy chain CDRs of CDR-H1, CDR-H2 and CDR-H3, above,respectively. Thus, in certain embodiments, a binding protein orantibody of this invention comprises or consists of CDR-H1 (SEQ ID NO:42), CDR-H2 (SEQ ID NO: 43) or CDR-H3 (SEQ ID NO: 44), and CDR-L1 (SEQID NO: 45), CDR-L2 (SEQ ID NO: 46) or CDR-L3 (SEQ ID NO: 47).

In certain embodiments, this invention provides a CD154 binding protein,e.g., an anti-CD154 antibody, wherein the binding protein or antibodycomprises or consists of the following three CDR L sequences: CDR-L1(SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47), andwherein the binding protein or antibody further comprises or consists ofthe following three CDR H sequences: CDR-H1 (SEQ ID NO: 42), CDR-H2 (SEQID NO: 43) and CDR-H3 (SEQ ID NO: 44).

In further embodiments, this invention provides a binding protein, e.g.,antibody, that specifically binds CD154 and which comprises or consistsof a variable light chain (V_(L)) sequence of SEQ ID NO: 54. Theinvention also relates to a CD154 binding protein or anti-CD154 antibodythat comprises or consists of a variable heavy chain (V_(H)) sequence ofSEQ ID NO: 56. In certain embodiments, a CD154 binding protein oranti-CD154 antibody of the invention may comprise or consist of both aV_(L) sequence of SEQ ID NO: 54 and a V_(H) sequence of SEQ ID NO: 56.

In other embodiments, the present invention relates to a CD154 bindingprotein, e.g., an anti-CD154 antibody, that specifically binds CD154,wherein the CD154 binding protein or anti-CD154 antibody comprises orconsists of one or more heavy chain (H) CDR sequence(s) selected fromCDR-H1 (SEQ ID NO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO:50). In further embodiments, the CD154 binding protein or anti-CD154antibody comprises or consists of at least two CDRs selected from CDR-H1(SEQ ID NO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50). Inyet further embodiments, the CD154 binding protein or anti-CD154antibody comprises or consists of all three CDR H sequences, which areCDR-H1 (SEQ ID NO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO:50).

In certain embodiments, this invention provides a CD154 binding protein,e.g., an anti-CD154 antibody, that specifically binds a CD154 protein,wherein the CD154 binding protein or anti-CD154 antibody comprises orconsists of one or more CDR light chain (L) sequence(s) selected fromCDR-L1 (SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3 (SEQ ID NO:53). In further embodiments, the CD154 binding protein or anti-CD154antibody comprises or consists of at least two CDRs selected from CDR-L1(SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3 (SEQ ID NO: 53). Inyet further embodiments, the CD154 binding protein or anti-CD154antibody comprises or consists of all three CDR L sequences, which areCDR-L1 (SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3 (SEQ ID NO:53).

In certain embodiments, a CD154 binding protein, e.g., an anti-CD154antibody, of the present invention comprises a complementary sequencecomprising or consisting of one or more light chain CDRs of CDR-L1,CDR-L2 and CDR-L3, above, or a complementary sequence comprising orconsisting of one or more heavy chain CDRs of CDR-H1, CDR-H2 and CDR-H3,above, respectively. Thus, in certain embodiments, an antibody of thisinvention comprises or consists of a CDR H selected from CDR-H1 (SEQ IDNO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50), or a CDR Lselected from CDR-L1 (SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3(SEQ ID NO: 53).

In certain embodiments, this invention provides a CD154 binding protein,e.g., an anti-CD154 antibody, wherein the CD154 binding protein oranti-CD154 antibody comprises or consists of all three CDR L sequences,which are CDR-L1 (SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3 (SEQID NO: 53), and wherein the binding protein, e.g., antibody, furthercomprises or consists of all three CDR H sequences, which are: CDR-H1(SEQ ID NO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50).

In further embodiments, this invention provides a CD154 binding protein,e.g., an anti-CD154 antibody, that specifically binds CD154, wherein theCD154 binding protein or anti-CD154 antibody comprises or consists of aV_(L) sequence of SEQ ID NO: 58. In additional embodiments, the antibodycomprises or consists of a V_(H) sequence of SEQ ID NO: 60. In furtherembodiments, the antibody comprises or consists of a V_(H) sequence ofSEQ ID NO: 60 and a V_(L) sequence of SEQ ID NO: 58.

In certain embodiments, the CD154 binding protein of this inventioncomprises a light chain sequence according to SEQ ID NO: 62 and a heavychain sequence according to SEQ ID NO: 65. In other embodiments, theCD154 binding protein of this invention comprises a light chain sequenceaccording to SEQ ID NO: 63 and a heavy chain sequence according to SEQID NO: 66.

In certain embodiments, the CD154 binding protein of this inventioncomprises a light chain sequence according to SEQ ID NO: 68 and a heavychain sequence according to SEQ ID NO: 71. In other embodiments, theCD154 binding protein of this invention comprises a light chain sequenceaccording to SEQ ID NO: 69 and a heavy chain sequence according to SEQID NO: 72. In other embodiments, the CD154 binding protein comprises atleast one of the sequences according to SEQ ID NO: 68, SEQ ID NO: 69,SEQ ID NO: 71 and SEQ ID NO: 72.

The CDR sequences of SEQ ID NOS: 3-8 are derived from rat monoclonalantibody 342. In an alternative embodiment of the invention, theanti-CD154 antibody is the rat 342 antibody comprising the V_(H) domainsequence of SEQ ID NO: 29 and the V_(L) domain sequence of SEQ ID NO:30. The invention also provides an isolated, recombinant or syntheticDNA molecule that comprises or consists of at least one sequenceselected from SEQ ID NO: 31 and SEQ ID NO: 32. Additionally provided isa vector comprising at least one sequence selected from SEQ ID NO: 33and SEQ ID NO: 34.

The invention also provides CD154 binding proteins, e.g., anti-CD154antibodies, that bind selectively to the same epitope as does any one ofthe anti-CD154 antibodies disclosed herein (e.g., 342, 381 and 338antibodies and epitope binding sequences thereof). In particular,antibodies 342 and 338 of the present invention exhibit similar CD154binding properties when used as first or second antibodies incompetition assays with the anti-CD154 antibody 5c8, as describedherein.

Thus, in certain embodiments, the invention provides CD154 bindingproteins and anti-CD154 antibodies that bind to the same epitope as doesa humanized antibody comprising a heavy chain sequence according to SEQID NO. 12 or SEQ ID NO. 13 and comprising a light chain sequenceaccording to SEQ ID NO. 15 (342 Fab and Fab′ fragments), and whichexhibit similar CD154 binding properties when used as first or secondantibodies in competition assays with the anti-CD154 antibody 5c8, asdescribed herein. In other embodiments, the invention provides CD154binding proteins and anti-CD154 antibodies that bind to the same epitopeas does a humanized antibody comprising a V_(L) domain sequenceaccording to SEQ ID NO. 58 and a V_(H) domain sequence according to SEQID NO. 60 (338 antibody variable sequences), and which exhibit similarCD154 binding properties in competition assays with anti-CD154 antibody5c8, as described herein.

In any of the above embodiments relating to a CD154 binding protein ofthe invention, the binding protein may be PEGylated. In embodiments inwhich the CD154 binding protein is an anti-CD154 antibody, antibodypolypeptide or fragment or derivative thereof, the antibody may bePEGylated on the heavy chain, the light chain, or on both chains.

The present invention also provides an isolated, recombinant and/orsynthetic DNA molecule that comprises or consists of at least onesequence selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 andSEQ ID NO: 25.

In certain embodiments, this invention also provides an isolated,recombinant and/or synthetic DNA molecule that comprises or consists ofat least one sequence selected from SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 27, SEQ ID NO: 67, SEQ ID NO: 70 and SEQ ID NO: 73.

In other embodiments, the invention provides an isolated, recombinantand/or synthetic DNA molecule that comprises or consists of at least onesequence selected from SEQ ID NO: 28 and SEQ ID NO: 41.

This invention also provides a vector that comprises any one of theisolated, recombinant and/or synthetic DNA molecules of this invention.In one embodiment, the vector comprises at least one sequence selectedfrom SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQID NO: 41.

In additional embodiments, this invention provides an isolated,recombinant, and/or synthetic DNA molecule that comprises or consists ofat least one sequence selected from SEQ ID NO: 55 and SEQ ID NO: 57. Insome embodiments, the invention provides an isolated, recombinant,and/or synthetic DNA molecule comprising or consisting of both SEQ IDNO: 55 and SEQ ID NO: 57.

In additional embodiments, this invention provides an isolated,recombinant, and/or synthetic DNA molecule that comprises or consists ofat least one sequence selected from SEQ ID NO: 59 and SEQ ID NO: 61. Insome embodiments, the invention provides an isolated, recombinant,and/or synthetic DNA molecule comprising or consisting of both SEQ IDNO: 59 and SEQ ID NO: 61.

In any of the embodiments of the invention relating to CD154 bindingproteins and anti-CD154 antibodies that comprise sequences whichcontribute to effector functions, said binding proteins or anti-CD154antibodies may additionally be selected or engineered to elicit reducedeffector function compared to an anti-CD154 antibody having an Fcregion, as described elsewhere herein. For example, CD154 bindingproteins and anti-CD154 antibodies that are free of an Fc region orconstant region sequences, or which lack a functional Fc region orconstant region sequences, may be selected for use in the invention.

In certain embodiments, CD154 binding proteins and anti-CD154 antibodiesof the invention are monovalent for binding to CD154 and preferablyelicit reduced effector functions when administered to a subjectrelative to a comparable CD154 binding protein such as a bivalentanti-CD154 antibody.

In certain other embodiments, Fc or constant region sequences, ifpresent in a CD154 binding protein, e.g., anti-CD154 antibodypolypeptide, may be selected or engineered to comprise one or moremodifications (e.g., amino acid substitutions, insertions, adducts ordeletions) that reduce or eliminate one or more effector function(s)relative to a control anti-CD154 antibody comprising native, parental orunmodified Fc or constant region sequences.

In some embodiments of the invention, an Fc region, when present, is anFc region of or derived from an IgG1, IgG2, IgG3 or IgG4 antibody. Insome embodiments, hybrid Fc regions may be used, i.e., IgG1/IgG4 hybridFc sequences. In particular embodiments, the Fc region comprises IgG4 Fcsequences or is derived from an IgG4 antibody. It is to be understoodthat any hybrid combination between different Fc regions that reducesone or more effector functions may be used according to the invention.

In certain other embodiments, glycosylation of the Fc portion of anantibody is reduced or eliminated, or the glycosylation profile of theantibody altered, as described further herein. In certain embodiments, aCD154 binding protein or anti-CD154 antibody polypeptide comprises aC_(H2) domain with an Fc region having a modification at or close to theconserved N-linked glycosylation site. The modification at the conservedN-linked glycosylation site may comprise a mutation in or near the heavychain glycosylation site, wherein the mutation reduces, alters orprevents glycosylation at the site. In further embodiments, themodification comprises mutation N298Q (N297 using EU Kabat numbering).In certain embodiments, the modification comprises removal of C_(H2)domain glycans or portions thereof. In certain alternative embodiments,the modification prevents formation of a mature N-glycan at theglycosylation site.

In some embodiments, the present invention relates to a CD154 bindingprotein, e.g., an anti-CD154 antibody, comprising a heavy chain CDR3sequence selected from SEQ ID NOS: 5, 44 and 50, and a variant Fcregion, the variant Fc region comprising a first amino acid residue andan N-glycosylation site, the first amino acid residue modified with sidechain chemistry or by amino acid substitution to achieve increasedsteric bulk or increased electrostatic charge compared to the unmodifiedfirst amino acid residue, thereby reducing the level of or otherwisealtering glycosylation at the N-glycosylation site. In certain of theseembodiments, the variant Fc region confers reduced effector functioncompared to a control, non-variant Fc region.

In certain embodiments, the invention relates to a CD154 bindingprotein, e.g., an anti-CD154 antibody, comprising a heavy chain CDR3sequence selected from SEQ ID NOS: 5, 44 and 50, and a variant Fcregion, the variant Fc region comprising a first amino acid residue andan N-glycosylation site, the first amino acid residue comprising acysteine thiol thereby reducing the level of or altering glycosylationat the N-glycosylation site, wherein the variant Fc region confersreduced effector function.

In certain embodiments, the first amino acid residue and theN-glycosylation site of the anti-CD154 antibodies comprising variant Fcregions described above are near or within an N-linked glycosylationmotif. In further embodiments, the N-linked glycosylation motifcomprises the amino acid sequence NXT or NXS. In certain embodiments,the N-linked glycosylation motif comprises the amino acid sequence NXT.In certain embodiments, the N-glycosylation site is located at aminoacid 297 according to the Kabat numbering system. In additionalembodiments, the modified first amino acid residue is amino acid 299according to the Kabat numbering system.

In certain of the above embodiments, the reduced effector functionexhibited by any one of the antibodies or antibody fragments containingbinding proteins described herein is reduced binding to an Fc receptor(FcR). In certain embodiments, the Fc receptor (FcR) is selected fromFcγRI, FcγRII and FcγRIII, and one or more subtypes thereof, such asFcγRIIa. In some embodiments, the FcR binding is reduced by a factor ofat least about 1.5-fold or more, about 2-fold or more, about 3-fold ormore, about 4-fold or more, about 5-fold or more, about 6-fold or more,about 7-fold or more, about 8-fold or more, about 9-fold or more, about10-fold or more, about 15-fold or more, about 50-fold or more, or about100-fold or more.

In certain embodiments, a CD154 binding protein, e.g., an anti-CD154antibody, of the invention with one or more reduced effector function(s)as described herein does not bind to a specific effector receptor oreffector receptor subtype. In certain of these embodiments, the CD154binding protein, e.g., an anti-CD154 antibody, does not bind to FcγRIIa.

In certain embodiments, the reduced effector function exhibited by anyone of the antibodies described herein is reduced binding to acomplement protein. In some embodiments, the complement protein is C1q.In certain embodiments, the reduced binding to a complement protein isby a factor of about 1.5-fold or more, about 2-fold or more, about3-fold or more, about 4-fold or more, about 5-fold or more, about 6-foldor more, about 7-fold or more, about 8-fold or more, about 9-fold ormore, about 10-fold or more, or about 15-fold or more.

In further embodiments of the present invention, a CD154 bindingprotein, e.g., an anti-CD154 antibody, of the invention comprises one ormore modifications or variations in the Fc region, is not glycosylatedand elicits one or more reduced effector functions when administered toa subject.

In certain embodiments, a CD154 binding protein, e.g., an anti-CD154antibody, of the invention causes fewer thromboembolic effects than doesadministration of anti-CD154 antibody 5c8 when administered to asubject.

In certain embodiments of the present invention, the modified firstamino acid residue of the CD154 binding proteins or anti-CD154antibodies comprising variant Fc regions described above is linked to afunctional moiety. In further embodiments, the functional moiety is ablocking moiety, a detectable moiety, a diagnostic moiety or atherapeutic moiety, or a combination thereof. A blocking moiety, incertain embodiments, may be, for example, a cysteine adduct, mixeddisulfide, polyethylene glycol or polyethylene glycol maleimide. Adetectable moiety, in certain embodiments, may be, for example, afluorescent moiety, a luminescent moiety or an isotopic moiety. Inembodiments where a diagnostic moiety is used, the diagnostic moiety maybe capable of revealing the presence of a condition, disease ordisorder. In other embodiments, a therapeutic moiety such as ananti-inflammatory agent, an anti-cancer agent, an anti-neurodegenerativeagent, an antibody that is selective for a molecule other than CD154, oran anti-infective agent may be used.

In certain embodiments of the present invention, a CD154 bindingprotein, e.g., an anti-CD154 antibody, comprises a modified amino acidresidue, the modification allowing site-directed conjugation of theprotein or antibody to a functional moiety, such as for site-directedpegylation. In certain embodiments, the modified amino acid residue is acysteine residue modified by a cysteine or mixed disulfide adduct. Thus,in certain embodiments, the CD154 binding protein is an anti-CD154antibody polypeptide that is pegylated at a modified amino acid residue,e.g., at a cysteine or lysine residue. In certain embodiments, theanti-CD154 antibody is a Fab or Fab′ fragment pegylated withPEG-maleimide.

This invention also provides nucleic acid, e.g., DNA sequences encodingthe CD154 binding proteins, e.g., anti-CD154 antibodies, of theinvention. The DNA sequences of the present invention may comprisesynthetic DNA, for instance produced by chemical processing, cDNA,genomic DNA or any combination thereof. This invention further providescloning or expression vectors comprising one or more nucleic acid, e.g.,DNA sequences of the present invention. The invention also relates, insome embodiments, to a vector comprising any of the synthetic, isolated,and/or recombinant nucleic acids described above.

This invention also provides a host cell comprising a DNA sequence orvector of the invention. In certain aspects, the present inventionrelates to a method for producing a CD154 binding protein, e.g., ananti-CD154 antibody, comprising culturing a host cell comprising any ofthe vectors described above under conditions suitable for producing theCD154 binding protein or anti-CD154 antibody by the host cell. In someembodiments, the method comprises recovering the CD154 binding proteinor anti-CD154 antibody from the host cell culture.

In certain embodiments, the CD154 binding protein, anti-CD154 antibodyor nucleic acid of this invention is labeled with a detectable marker,which may be a radioactive isotope, enzyme, dye or biotin. In certainother embodiments, the antibody of this invention is conjugated to atleast one other therapeutic agent, which may be a radioisotope,radionuclide, toxin, toxoid, a non-CD154 specific antibody polypeptideor fragment (i.e., creating a bispecific or multispecific antibody), orchemotherapeutic agent, for example. In yet other embodiments, theantibody of this invention is conjugated to an imaging agent, whichcould be a labeling moiety. The labeling agent may also be a biotin, afluorescent or luminescent moiety, a radioactive moiety, a histidinetag, or a peptide tag.

The present invention also relates to sequence variants of the CD154binding proteins, e.g., anti-CD154 antibodies, described herein, andnucleic acid sequences that encode them. Sequence variants of theinvention preferably share at least 90%, 91%, 92%, 93% or 94% identitywith a polypeptide of the invention or with a nucleic acid sequence thatencodes it. More preferably, a sequence variant shares at least 95%,96%, 97% or 98% identity at the amino acid or nucleic acid level. Mostpreferably, a sequence variant shares at least 99%, 99.5%, 99.9% or moreidentity with a polypeptide of the invention or a nucleic acid sequencethat encodes it.

Accordingly, the present invention provides an isolated proteincomprising a sequence that is at least 80, 85, 90, 92, 94, 95, 96, 97,98, 99, 99.5, 99.9 or 100% identical to the sequence of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:68, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 72.

In certain embodiments, a chimeric, humanized or human anti-CD154antibody polypeptide of the invention is, for example, a dAb, Fab, Fab′,scFv, Fv, a disulfide-bonded Fv or comprises a single immunoglobulinvariable domain, such as a V_(H) or a V_(L) domain, that is specific andmonovalent for CD154 binding. Certain CD154 binding proteins, e.g.,chimeric, humanized or human anti-CD154 antibody polypeptides, of theinvention comprise one, two or more CDRs of the invention andalternative scaffold or universal framework sequences. In certainembodiments, CD154 binding proteins and anti-CD154 antibodies of theinvention comprise a single variable domain selected from a variableheavy chain (V_(H)) and a variable light chain (V_(L)).

This invention also provides a CD154 binding protein, e.g., a chimeric,humanized or human anti-CD154 antibody polypeptide, that is monovalentfor binding to CD154 which is conjugated to a functional moiety thatincreases its in vivo half-life relative to the same polypeptide lackingthe functional moiety. In certain embodiments, the functional moietycomprises or consists of polyethylene glycol. In certain embodiments,the functional moiety comprises or consists of an albumin molecule, suchas human serum albumin. Accordingly, the invention provides a PEG-linkedCD154 binding protein, e.g., a PEG-linked chimeric, humanized or humananti-CD154 antibody polypeptide that specifically and monovalently bindsCD154, and which has an increased in vivo half-life relative to the samepolypeptide lacking linked polyethylene glycol.

This invention also provides a pharmaceutical composition comprising atleast one CD154 binding protein, e.g., an anti-CD154 antibody, of thepresent invention, which may, in some embodiments, further comprise apharmaceutically acceptable carrier. A pharmaceutical composition of theinvention may optionally further comprise an additional bioactive ortherapeutic agent, such as an immunosuppressive or immunomodulatorycompound or agent, or a diagnostic agent. In certain embodiments, thecomposition comprises a therapeutically effective amount of a pegylated,anti-CD154 Fab′ antibody having the epitope specificity of anti-CD154antibody 342 or 338, as described herein.

The present invention further provides a method for treating orpreventing a human condition, disorder or disease mediated in whole orin part by CD40 signaling, or a symptom of any of the foregoing, themethod comprising the step of administering to a subject in need thereofa therapeutically effective amount of a pharmaceutical compositioncomprising a CD154 binding protein, e.g., an anti-CD154 antibody, of thepresent invention, such that therapy or prevention of the condition,disease or disorder is achieved.

This invention also provides a method for inhibiting or preventing oneor more of an immune, auto-immune or inflammatory response mediated inwhole or in part by CD40 signaling, or a symptom of any of theforegoing, the method comprising the step of administering to a subjectin need thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a CD154 binding protein, e.g., an anti-CD154antibody, of the present invention, such that a therapeutic orpreventive response is achieved. In certain embodiments, the method isused to treat a subject presenting signs of or diagnosed with systemiclupus erythematosus (SLE). In other embodiments, the method is used totreat a subject presenting signs of or diagnosed with a rheumatoidcondition, such as rheumatoid arthritis (RA).

This invention provides a human antibody polypeptide that is monovalentfor binding to CD154. In certain embodiments, the human antibodypolypeptide that is monovalent for binding to CD154, when present at orabove saturating concentrations for CD154 binding based on its bindingaffinity, both blocks binding of antibody 5c8 to CD154 when added toCD154 first, and displaces antibody 5c8 bound to CD154 when added afterantibody 5c8 is added. In certain embodiments, the human antibodypolypeptide is PEG-linked. In certain embodiments, the human antibodypolypeptide is free of an Fc domain. In certain of the aboveembodiments, the CD154 binding protein or human antibody polypeptidedoes not displace CD154 bound to CD40.

In certain embodiments, the invention provides a pegylated anti-CD154Fab′ antibody that is monovalent for CD154 binding and further providesmethods for treating or preventing one or more symptoms of arthritis,such as rheumatoid arthritis, or systemic lupus erythematosus (SLE),comprising the step of administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising the pegylated, anti-CD154 Fab′ antibody, such that atherapeutic or preventive response is achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table providing the amino acid sequences for thecomplementarity determining regions (CDRs) of the anti-CD154 antibodies342, 381, and 338.

FIG. 2 is a table providing the amino acid and corresponding nucleotidesequences for the V_(H) and V_(L) domains of the rat anti-CD154 antibody342. Underlined sequences correspond to the nucleotide sequence encodinga leader (i.e., signal) peptide.

FIG. 3 is a table providing the amino acid and corresponding nucleotidesequences of human acceptor frameworks used to produce humanizedanti-CD154 antibodies in the Examples.

FIG. 4 is a table providing the amino acid and corresponding nucleotidesequences of V_(H) and V_(L) domains in which the 342 CDRs have beengrafted into the frameworks shown in FIG. 3.

FIG. 5 is a table providing the amino acid sequences and correspondingnucleotide sequences of V_(H) domains of antibody 342 (VH3 gH1 and VH4gH1) in which the 342 CDRs were grafted into human acceptor frameworksand wherein certain key donor residues were maintained.

FIG. 6 is a table providing the amino acid and corresponding nucleotidesequences of a V_(L) domain of antibody 342 (VK1 gL4) in which the 342CDRs were grafted into human acceptor frameworks and wherein certain keydonor residues were maintained.

FIG. 7 is a table providing sequences for the complete light chain(variable and constant light chain regions) and heavy chain regions ofantibody 342. Sequences corresponding to signal/leader peptides areunderlined.

FIG. 8 shows the nucleotide sequence of expression inserts that wereused to make Fab (SEQ ID NO: 28) and Fab′ (SEQ ID NO: 41) versions ofgraft 342 gL4gH1. The signal/leader sequences are underlined andrestriction sites (used for cloning into the E. coli expression vectoras described in Example 2) are capitalized and shown in bold.

FIG. 9 shows an alignment of light and heavy chains of the rat 342anti-CD154 antibody (donor) amino acid sequence with the human germline(acceptor) frameworks used in the humanization of the 342 antibody.“Light 342” is the rat V_(L) domain sequence. “Heavy 342” is the ratV_(H) domain sequence. The CDR residues are bold and underlined. Theacceptor framework light (2 1 1 O12; SEQ ID NO: 35) and heavy (1-1 3-66;SEQ ID NO: 37 and 1-1 4-59; SEQ ID NO: 39) chains are shown. CDR-onlygrafts into human germline acceptor frameworks are also shown (VK1 gL3,VH3 gH7 and VH4 gH6). In certain humanized heavy and light chains, donorresidues of the 342 antibody are retained within the framework region,and these key donor framework residues are shown in bold italics and arehighlighted. The VK1 gL4 donor content is R38, Y71 and S85. The VH3 gH1donor content is V24, M48, G49, T73 and V78. The VH4 gH1 donor contentis M48, R71 and V78. SEQ ID NOs for the light chains shown, in orderfrom top to bottom are SEQ ID NO: 30 (“342”), SEQ ID NO: 35 (“2 1 1O12”), SEQ ID NO: 14 (“342 gL3”) and SEQ ID NO: 2 (“342 gL4”). SEQ IDNOs for the heavy chains shown (VH3 grafts), in order from top tobottom, are SEQ ID NO: 29 (“342”), SEQ ID NO: 37 (“1-1 3-66”), SEQ IDNO: 10 (“342 gH7”), and SEQ ID NO: 1 (“342 gH1”). SEQ ID NOs for theheavy chains shown (VH4 grafts), in order from top to bottom, are SEQ IDNO: 29 (“342”), SEQ ID NO: 39 (“1-1 4-59”), a variant of SEQ ID NO: 9(“VH4 gH6”), and a variant of SEQ ID NO: 11 (“VH4 gH1”). The SEQ ID NO:9 and SEQ ID NO: 11 variants shown in FIG. 9 begin with amino acid “E”(instead of “Q” as in FIGS. 4 and 5, respectively) for expression in E.coli. Accordingly, the nucleotide sequences that correspond to thesevariant SEQ ID NOs: 9 and 11 differ from the nucleotide sequences setforth in SEQ ID NO: 19 and SEQ ID NO: 21 in that they begin withnucleotide “g” instead of “c”.

FIG. 10 provides V_(L) and V_(H) amino acid and corresponding nucleotidesequences for anti-CD154 antibody 381. The CDR amino acid sequences areunderlined.

FIG. 11 provides V_(L) and V_(H) amino acid and corresponding nucleotidesequences for anti-CD154 antibody 338. The CDR amino acid sequences areunderlined.

FIG. 12 is a table listing rat anti-human CD154 antibodies isolated bySelected Lymphocyte Antibody Method (SLAM). The table provides K_(d) andIC₅₀ values obtained with these antibodies in BIACORE® (an assay tomeasure molecular interactions), CD40 binding assays and ICAM-1upregulation assays. Data obtained with antibody 342 are highlighted.

FIG. 13 provides the kappa light chain amino acid and correspondingnucleotide sequences of aglycosylated anti-CD154 antibody hu5c8 (hu5c8aglyP-huIgG4).

FIG. 14 provides the heavy chain amino acid and corresponding nucleotidesequences of aglycosylated anti-CD154 antibody hu5c8 (hu5c8aglyP-huIgG4). The mutations made to render the variant aglycosylated(S228P/T299A in Kabat EU nomenclature; residues 226 and 297) are shownunderlined and in bold in the mature protein sequence.

FIG. 15 provides the kappa light chain amino acid and correspondingnucleotide sequences of aglycosylated anti-CD154 antibody hu342 (hu342aglyP-huIgG4).

FIG. 16 provides the heavy chain amino acid and corresponding nucleotidesequences of aglycosylated anti-CD154 antibody hu342 (hu342aglyP-huIgG4). The mutations made to render the variant aglycosylated(S228P/T299A in Kabat EU nomenclature; residues 226 and 297) are shownunderlined and in bold in the mature protein sequence.

FIG. 17 shows exemplary Fab′ fragments and a gel showing thesite-specific PEGylation of an Fab′ fragment of an anti-CD154 antibody.The Fab′ was pegylated by reacting PEG-maleimide with a single cysteineresidue at the hinge.

FIG. 18 is a table providing K_(d) and IC₅₀ values obtained in BIACORE®(an assay to measure molecular interactions), CD40 binding assays,ICAM-1 upregulation assays and CD40L competition binding assays fordifferent embodiments of the humanized 342 gL4gH1 antibody, includingFab′ fragments and antibody-PEG conjugates.

FIG. 19 shows two graphs of the anti-TT (tetanus toxoid) IgG titervalues as a function of time in cynomolgus monkeys receiving either asaline control or various doses of anti-CD154 antibodies. The top graphshows IgG titer values for days 0-20 after antibody treatment and TTchallenge (the primary immune response), and the bottom graph showsvalues for days 30-50 after antibody treatment and a second TT challengeon day 30.

FIG. 20 is a graph of the anti-TT (tetanus toxoid) IgG titer values as afunction of time in cynomolgus monkeys receiving various formats ofanti-CD154 antibodies at a single dose (20 mg/kg for hu5c8, aglycosyl5c8 and aglycosyl 342 and 40 mg/kg for 342 Fab′-PEG and 342 DFM-PEG).DFM-PEG is an antibody fragment in which two Fab′ fragments arecross-linked with a PEGylated dimaleimide bridge.

FIG. 21 is a graph of the anti-TT (tetanus toxoid) IgM titer values as afunction of time in cynomolgus monkeys receiving either a saline controlor various doses of anti-CD154 antibodies. The graph shows IgM titervalues for days 0-20 after challenge with TT (primary response).

FIG. 22 is a graph comparing the pharmacokinetics in cynomolgus monkeysof the 342 Fab′-PEG antibody and hu5c8 antibody.

FIGS. 23A-23H show the results of flow cytometry analysis ofcross-blocking of labeled 342 Fab′ binding to CD154-expressing D1.1Jurkat cells pre-bound with an unlabeled first anti-CD154 Fab′, asindicated in each panel (23A—A33; 23B—338; 23C—402; 23D—381; 23E—300;23F—294; 23G—335; 23H—303) (see Example 9). A33 is an isotype-matchedcontrol antibody and confirms that there is no non-specificcross-blocking.

FIGS. 24A-24H show the results of flow cytometry analysis ofcross-blocking of labeled Hu5c8 Fab′ binding to CD154-expressing D1.1Jurkat cells pre-bound with an unlabeled first anti-CD154 Fab′, asindicated in each panel (24A—338; 24B—402; 24C—381; 24D—303; 24E—335;24F—300; 24G—294; 24H—A33) (see Example 9). A33 is an isotype-matchedcontrol antibody and confirms that there is no non-specificcross-blocking.

FIGS. 25A-25F show the results of BIACORE® (an assay to measuremolecular interactions) analysis of competitive binding of various formsof the 342 and Hu5c8 antibodies to soluble CD154 protein (ECD) or aCD40:CD154 complex. FL—full length. CD40hFc-human CD40 fusion protein.

FIG. 26 is a graph showing the results of a platelet aggregation assaydescribed in Example 11 (Assay 1) performed with the Hu5c8 anti-CD154antibody, 342 Fab′-PEG anti-CD154 antibody, and control antibodies. Thefirst panel shows results obtained with CD40L complexes formed withwhole IgG and the second panel shows complexes formed with Fab′-PEG ordiFab′-PEG. The third panel shows results obtained with triFab′-PEG andaglycosylated anti-CD154 antibody hu342.

FIG. 27 is a graph showing the results of a platelet aggregation assaydescribed in Example 11 (Assay 2) performed with positive and negativecontrol antibodies. The results demonstrate that platelet aggregation isspecifically enhanced by complexes of the positive control anti-CD154antibody and recombinant human soluble CD40L (sCD154).

FIG. 28 shows a sequence alignment of human (SEQ ID NO: 76) and mouse(SEQ ID NO: 77) soluble CD40L amino acid sequences. Differences betweenhuman and mouse sequences are indicated in red. The Hu5c8 epitopesequences are indicated in blue. Internal residues are marked with “|”.Six regions (1-6) of the sequence where human residues were introducedinto soluble mouse CD40L were selected.

FIGS. 29A-29D show the results of a competition ELISA assay todemonstrate cross-blocking of 342 Fab′ and hu5c8 Fab′ (see Example 14).FIG. 29A shows the results of a titration of biotin 342 Fab′ on CD154. A1 nM concentration is on the linear part of the curve. FIG. 29B showsthe results of a titration of biotin hu5c8 Fab′ on CD154. A 0.3 nMconcentration is on the linear part of the curve. FIG. 29C showscross-blocking of biotin 342 and biotin 5c8 by unlabeled 342 Fab′. ch342Fab is the chimeric 342 Fab′. FIG. 29D shows cross-blocking of biotin342 by unlabeled 5c8 Fab′.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention pertains. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.

Throughout this application, various patents, publications andreferences are referred to. Disclosures of these patents, publicationsand references are hereby incorporated by reference into thisapplication in their entireties.

Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al., Current Protocols In Molecular Biology, John Wiley &Sons, New York (1998 and Supplements to 2001); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds., HandbookOf Molecular And Cellular Methods In Biology And Medicine, CRC Press,Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A PracticalApproach, IRL Press, Oxford (1991).

Standard reference works setting forth the general principles ofimmunology known to those of skill in the art include Harlow and Lane,Antibodies: A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1999), and Roitt et al., Immunology, 3dEd., Mosby-Year Book Europe Limited, London (1993). Standard referenceworks setting forth the general principles of medical physiology andpharmacology known to those of skill in the art include Fauci et al.,Eds., Harrison's Principles Of Internal Medicine, 14th Ed., McGraw-HillCompanies, Inc. (1998).

Definitions

As used herein, the term “CD154 binding protein” includes any molecule,including an antibody, that specifically binds to or antagonizes CD154.Thus, as used herein, an anti-CD154 antibody is one class of CD154specific binding proteins. A CD154 binding protein of the invention maycomprise at least one, preferably two, three or more CDRs as disclosedherein. A CD154 binding protein or other such CD154 antagonist mayencompass species that are not classic antibody fragments or derivativesbut which nonetheless comprise amino acid sequences and/or chemicalstructures that confer CD154 epitope binding specificity. Such CD154antagonists may be made, e.g., from alternative scaffolds (see, forexample, Binz et al. 2005 Nat Biotech 23: 1257-1268 and Hosse et al.2006 Protein Science 15: 14-27). Such a CD154 binding protein orantagonist may be fused to an antibody Fc region that is functionallydeficient or to a heterologous functional moiety as described herein toimprove the half-life and/or other in vivo properties of the CD154binding protein.

The CD154 binding proteins may comprise at least one of the CDRsdescribed herein incorporated into a biocompatible framework structure.In one example, the biocompatible framework structure comprises apolypeptide or portion thereof that is sufficient to form aconformationally stable structural support, framework, or scaffold,which is able to display one or more sequences of amino acids that bindto an antigen (e.g., CDRs, a variable domain, etc.) in a localizedsurface region. Such structures can be a naturally occurring polypeptideor polypeptide “fold” (a structural motif), or can have one or moremodifications, such as additions, deletions or substitutions of aminoacids, relative to a naturally occurring polypeptide or fold. Thesescaffolds can be derived from a polypeptide of any species (or of morethan one species), such as a human, other mammal, other vertebrate,invertebrate, plant, bacteria or virus.

Typically the biocompatible framework structures are based on proteinscaffolds or skeletons other than immunoglobulin domains. For example,those based on fibronectin, ankyrin, lipocalin, neocarzinostain,cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-Dl, Z domain andtendramisat domains may be used (See, e.g., Nygren and Uhlen, 1997,Current Opinion in Structural Biology, 7, 463-469).

The term “antibody” as it is used herein with respect to the inventionincludes an isolated, recombinant or synthetic antibody, antibodyconjugate or antibody derivative. The term “antibody” is often intendedto include an antibody fragment, including an antigen-binding fragment,unless otherwise indicated or understood by context. An antigen-bindingfragment competes with the intact antibody for specific binding. Seegenerally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)). Antigen-binding fragments may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. In some embodiments, antigen-binding fragmentsinclude Fab, F(ab)₂, Fab′, F(ab′)₂, F(ab′)₃, Fd, Fv, domain antibodies(dAb), other monovalent and divalent fragments, complementaritydetermining region (CDR) fragments, single-chain antibodies (e.g., scFv,scFab, and scFabΔC), chimeric antibodies, diabodies, triabodies,minibodies, nanobodies, and polypeptides that contain at least a portionof an antibody that is sufficient to confer specific antigen binding tothe polypeptide, and fusions and derivatives of the foregoing. See,e.g., Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005) andHust et al., BMC Biotech 7: 14 (2007).

An “Fd fragment” is an antibody fragment that consists of the V_(H) andC_(H1) domains; an “Fv fragment” consists of the V_(L) and V_(H) domainsof a single arm of an antibody; an “scFv fragment” is a single chainantibody comprising a heavy chain variable region (V_(H)) and a lightchain variable region (V_(L)) joined by a peptide linker; an “scFabfragment” is a single chain antibody comprising a fragment difficult(Fd) joined to a light chain by a peptide linker; an “scFabΔC” fragmentis an scFab variant without cysteine (see, e.g., Hust et al., supra);and a “dAb fragment” (single domain antibody) comprises a singlevariable domain (e.g., a V_(H) or a V_(L) domain) (Ward et al., Nature341:544-546 (1989)). Further, diabodies and triabodies are alsoincluded. Diabodies and triabodies of the present invention include, forexample, homodimeric and heterodimeric diabodies and triabodies. Forexample, in certain embodiments, the variable domains making up atriabody may bind to three different epitopes or to identical epitopes.

Unless otherwise stated or where otherwise implied by context, an“antibody” of the present invention includes whole antibodies and anyantigen-binding fragments thereof, antibody derivatives or variants thatmay contain one or more modifications (e.g., an amino acid insertion,deletion, substitution, a post-translational modification or lackthereof, etc.), including antibody conjugates (i.e., antibody orantigen-binding fragment thereof conjugated to or associated with afunctional moiety). The antibody derivatives, including antibodyconjugates, may be based on or may comprise an antigen-binding fragmentof the invention that specifically binds CD154. Additionally, theaforementioned antibody embodiments may be murine, hamster, goat,rabbit, chimeric, humanized, or fully human antibodies, fragments,derivatives, or conjugates. It is understood that in certain aspects ofthe invention, the term “antibody” may exclude one or more of theantibody embodiments recited above; such conditions will be evident tothe skilled artisan.

The term “pegylation,” “polyethylene glycol” or “PEG” includes apolyalkylene glycol compound or a derivative thereof, with or withoutcoupling agents or derivatization with coupling or activating moieties(e.g., with thiol, triflate, tresylate, azirdine, oxirane, or preferablywith a maleimide moiety, e.g., PEG-maleimide). Other appropriatepolyalkylene glycol compounds include, but are not limited to, maleimidomonomethoxy PEG, activated PEG polypropylene glycol, and charged orneutral polymers of the following types: dextran, colominic acids, orother carbohydrate based polymers, polymers of amino acids, and biotinand other affinity reagent derivatives.

The term “effector function” refers to the functional ability of the Fcor constant region of an antibody to bind proteins and/or cells of theimmune system. Antibodies having reduced effector function and methodsfor engineering such antibodies are well-known in the art (see, e.g., WO05/18572, WO 05/03175, and U.S. Pat. No. 6,242,195) and are described infurther detail herein. Typical effector functions include the ability tobind complement protein (e.g., the complement protein C1q), and/or an Fcreceptor (FcR) (e.g., FcγRI, FcγRII, FcγRIIa, FcγRIII, and/or FcγRIIIb).The functional consequences of being able to bind one or more of theforegoing molecules include, without limitation, opsonization,phagocytosis, antigen-dependent cellular cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC) and/or effector cell modulation.A decrease in effector function refers to a decrease in one or more ofthe biochemical or cellular activities induced at least in part bybinding of Fc to its cognate receptor or to a complement protein oreffector cell, while maintaining the antigen-binding activity of thevariable region of the antibody (or fragment thereof), albeit withreduced, similar, identical, or increased binding affinity. Particularantibodies of the invention exhibit reduced effector function. Decreasesin effector function, e.g., Fc binding to an Fc receptor or complementprotein, can be expressed in terms of fold reduction (e.g., reduced by1.5-fold, 2-fold, and the like) and may be calculated based on, e.g.,the percent reductions in binding activity determined using bindingassays known in the art (see, for example, WO 05/18572).

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

CD154

CD154 is known by several other names in the art, such as CD40 ligand(CD40L), CD40 counter receptor (CD40CR), gp39, T-BAM, T-Cell ActivatingMolecule, TRAF, TNF-Related Activation Protein (TRAP), and TumorNecrosis Factor Ligand Superfamily Member 5 (TNFSF5) (Gauchat et al.,1993 FEBS Lett. 315: 259-266; Graf et al., 1992, Europ. J. Immun. 22:3191-3194; Hollenbaugh et al., 1992 EMBO J. 11: 4313-4321). These termsare used interchangeably throughout this application. The CD154 bindingproteins, including antibodies, of this invention specifically bind tohuman CD154 and may cross react and therefore specifically bind to CD154of other species. In certain embodiments, the CD154 binding proteins,including antibodies, of this invention specifically bind to humanCD154, mouse CD154 or non-human primate CD154.

Anti-CD154 Antibodies and CD154 Binding Proteins

The term “anti-CD154 antibody” as used herein refers to animmunoglobulin molecule that is able to bind specifically to an epitopeon a CD154 antigen. Anti-CD154 antibodies may be intact immunoglobulinsderived from natural sources or from recombinant sources and may beimmunoreactive portions of intact immunoglobulins. Antibodies aretypically tetramers of immunoglobulin molecules.

Accordingly, as referred to in all of the embodiments and methods ofthis invention an “anti-CD154 antibody” encompasses (unless whereotherwise indicated or where otherwise suggested by context) amonoclonal antibody, a polyclonal antibody, a murine antibody, hamsterantibody, goat antibody, rabbit antibody, a chimeric antibody, aprimatized antibody, a humanized antibody, a (fully) human antibody, amultimeric antibody, a heterodimeric antibody, a hemidimeric antibody, abi-, tri-, or tetravalent antibody, a bispecific antibody, a singlechain antibody (e.g., scFv, scFab, and scFabΔC), Bis-scFv, a diabody,triabody or tetrabody, single domain antibodies, and modified Fabfragments. In certain embodiments, the anti-CD154 antibody is anantibody comprising only a single variable immunoglobulin domain.Accordingly, monovalent antibodies include antibodies that comprise onlyone immunoglobulin variable domain (i.e., a single light or heavyvariable chain) and that specifically bind to CD154. In addition,anti-CD154 antibodies of the invention may be monovalent, divalent, ormultivalent for CD154.

In certain embodiments, a CD154 binding protein, e.g., an anti-CD154antibody, with reduced effector function comprises any portion of ananti-CD154 antibody that is sufficient to maintain specific binding tothe CD154 antigen. For example, the antibody may comprise only a singlevariable immunoglobulin domain—a V_(H) or V_(L) domain.

Accordingly, in certain embodiments, a CD154 binding protein, e.g., ananti-CD154 antibody, is an antibody fragment. Antibody fragmentsinclude, for example, an Fab fragment, an F(ab)₂ fragment, an Fab′fragment, an F(ab′)₂ fragment, an F(ab′)₃, fragment, a single chain F(v)fragment or an F(v) fragment and epitope-binding fragments of any of theabove (see for example Holliger and Hudson, 2005, Nature Biotech.23(9):1126-1136). Antibody fragments of the invention are described inmore detail below.

In one example, the CD154 binding proteins or anti-CD154 antibodies areantibodies (e.g., antibodies of the IgG4 subtype) or fragments (e.g.,Fab′ fragments) which possess a native or a modified hinge region. Anumber of modified hinge regions have been described, for example, inU.S. Pat. No. 5,677,425, WO 99/15549, and WO 98/25971. In anotherexample, the antibodies of the invention are modified in their constantregions as those antibodies described in WO 05/003169, WO 05/003170 andWO 05/003171. Any of the aforementioned anti-CD154 antibodies, antibodyderivatives or antibody fragments may be used to form antibodyconjugates of the present invention. Any of the above antibodies,fragments, and conjugates may elicit reduced effector function comparedto a second anti-CD154 antibody.

The antibody molecules of the invention can be of any class (e.g. IgG,IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule. Theconstant region domains of the antibody, if present, may be selectedhaving regard to the proposed function of the antibody molecule. Forexample, the constant region domains may be human IgA, IgD, IgE, IgG orIgM domains. In particular, human IgG constant region domains may beused, especially IgG1, IgG2, IgG3, and IgG4. IgG2 and IgG4 isotypes maybe used in certain embodiments where the antibody molecule is intendedfor therapeutic uses for which reduced or eliminated antibody effectorfunctions are desired. Alternatively, IgG1 and IgG3 isotypes may be usedwhen the antibody molecule is intended for therapeutic purposes forwhich antibody effector functions are required.

In some embodiments, one or more of the CDRs of a CD154 binding protein,e.g., antibody, of the invention may be incorporated into one or moreimmunoglobulin domains, universal frameworks, protein scaffolds or otherbiocompatible framework structures based on protein scaffolds orskeletons other than immunoglobulin domains (Nygren & Uhlén, 1997, Curr.Opin. Struct. Biol. 7:463-469; Saragovi et al, 1992, Bio/Technology10:773-779; Skerra, 2000, J. Mol. Recognition 13:167-187). In certainembodiments, the CDRs of an anti-CD154 antibody are incorporated into auniversal framework (i.e., a framework which can be used to create thefull variability of functions, specificities, or properties which areoriginally sustained by a large collection of different frameworks, seeU.S. Pat. No. 6,300,064). In other embodiments, alternative scaffolds(see, for example, Binz et al. 2005 Nat Biotech 23: 1257-1268 and Hosseet al. 2006 Protein Science 15: 14-27) may be used to create CD154binding proteins of the invention.

The term “anti-CD154 antibody” also encompasses a synthetic antibody ora recombinant antibody that is generated using recombinant DNAtechnology, such as an antibody expressed by a bacteriophage. The term“anti-CD154 antibody” should also be construed to include an antibodythat has been generated by the synthesis of a DNA molecule encoding theantibody and which DNA molecule expresses an antibody protein, or anamino acid sequence specifying the antibody, wherein the DNA or aminoacid sequence has been obtained using synthetic DNA or amino acidsequence technology that is available and well known in the art.

In one embodiment, the invention provides an “anti-CD154 antibody” thatis a monoclonal antibody. A monoclonal antibody refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma (murine or human) method first described by Kohleret al., Nature, 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature, 352:624-628 (1991) andMarks et al., J. Mol. Biol., 222:581-597 (1991), for example. It is alsounderstood that certain embodiments of the present invention relate tocompositions comprising one or more different monoclonal antibodies thatspecifically bind CD154, i.e., a polyclonal antibody compositioncomprising a plurality of monoclonal antibodies with different epitopespecificities.

In another embodiment of the invention, an “anti-CD154 antibody” refersto an antibody that is a chimeric antibody, or an antibody derivative orconjugate or antigen-binding fragment thereof. Typically, chimericantibodies include the heavy and/or light chain variable regions,including both CDR and framework residues, of one species (typicallymouse) fused to constant regions of another species (typically human).These chimeric mouse/human antibodies contain approximately 75% humanand 25% mouse amino acid sequences. The human sequences represent theconstant regions of the antibody, while the mouse sequences representthe variable regions (and thus contain the antigen-binding sites) of theantibody.

In another embodiment, the CD154 binding proteins and anti-CD154antibodies of this invention include antibodies, antibody derivativesand antigen-binding fragments comprising a variable domain comprisingframework regions from one antibody and CDR regions from anotherantibody.

In a more specific embodiment, the CD154 binding proteins and anti-CD154antibodies of this invention include chimeric antibodies comprisingframework regions and CDR regions from different human antibodies.

Methods of making all of the chimeric antibodies described above arewell known to one of skill in the art. See, e.g., U.S. Pat. No.5,807,715; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA81(21):6851-5; Sharon et al. (1984) Nature 309(5966):364-7; Takeda etal. (1985) Nature 314(6010):452-4.

In certain embodiments of the present invention, an anti-CD154 antibodythat binds CD154 is generated by Selected Lymphocyte Antibody Method(SLAM) (Babcook et al., 1996, Proc. Natl. Acad. Sci, 93, 7843-7848; WO92/02551; de Wildt et al., 1997, J. Immunol. Methods, 207:61-67 and inLagerkvist et al., 1995, BioTechniques 18:862-869) which enables theisolation from any species of cells producing high affinity antibodiesduring in vivo immune responses. Other techniques include thosedescribed by de Wildt et al., 1997, 1 Immunol. Methods, 207:61-67 andLagerkvist et al., 1995, BioTechniques 18(5):862-869. The above methodsrely on the isolation of individual antibody-producing cells which arethen clonally expanded followed by screening for those clones whichproduce anti-CD154 antibodies followed by the subsequent identificationof the sequence of their variable heavy (V_(H)) and light (V_(L)) chaingenes. A particular screening method is detailed in WO 04/051268. Thus,B cells that are positive for antibodies to CD154 are isolated. The Bcells may be from human, mouse, rat, hamster, rabbit, goat, or othermammalian species. The antibody genes in these B cells may be cloned andexpressed in a host cell, e.g., by conventional recombinant DNAtechnology. In certain embodiments, the host cell is E. coli. Other hostcells are detailed below. The antibodies (which include antibodyfragments such as Fab′ fragments) expressed in these cells may bepurified by conventional means. If the antibodies are from a non-humansource, they may be humanized by conventional methods, such as bymutagenesis of their genes. The humanized antibodies may be subsequentlyexpressed in a host cell and may be purified.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, Nature, 1975,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today, 1983, 4, 72) and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, pp.77-96, Alan R. Liss, Inc., 1985). The methods for creating andmanufacturing recombinant antibodies are well known in the art (see forexample, U.S. Pat. Nos. 4,816,397; 6,331,415; Simmons et al., 2002,Journal of Immunological Methods, 263, 133-147; WO 92/02551; Orlandi etal., 1989, Proc. Natl. Acad. Sci. USA, 86, 3833; Riechmann et al., 1988,Nature, 322, 323; U.S. Pat. No. 5,585,089; WO91/09967; Mountain andAdair, 1992, Biotechnol. Genet. Eng. Rev, 10, 1-142; Verma et al., 1998,J. Immunol. Methods, 216:165-181; Holliger and Hudson, 2005, NatureBiotech. 23(9):1126-1136).

Antibodies of the present invention may also be generated using variousphage display methods known in the art which include those disclosed byBrinkman et al., 1995, 1 Immunol. Methods, 182:41-50; Ames et al., 1995,J. Immunol. Methods, 184, 177-186; Kettleborough et al., 1994, Eur. J.Immunol., 24, 952-958; Persic et al., 1997, Gene, 187, 9-18; and Burtonet al., 1994, Advances in Immunol., 57, 191-280; WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; and WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743; and 5,969,108.

Also, transgenic (e.g., genetically engineered) mice, or otherorganisms, including other mammals, may be used to produce bindingproteins and antibodies of this invention (see for example U.S. Pat. No.6,300,129). For example, it is known that mice engineered to replaceonly the variable regions of mouse immune loci (heavy chain V, D, and Jsegments, and light chain V and J segments) with corresponding humanvariable sequences can be used to produce large quantities of highaffinity antibodies with human variable sequences (see, e.g., U.S. Pat.Nos. 6,586,251; 6,596,541, and 7,105,348).

In another embodiment of this invention, an “anti-CD154 antibody” refersto an antibody, antibody derivative or conjugate, or antigen-bindingfragment that is primatized or humanized. Primatized and humanizedantibodies typically include heavy and/or light chain CDRs from a murineantibody grafted into a non-human primate or human antibody V regionframework, usually further comprising a human constant region. See,e.g., Riechmann et al. (1988) Nature 332:323-327; U.S. Pat. Nos.6,054,297; 5,821,337; 5,770,196; 5,766,886; 5,821,123; 5,869,619;6,180,377; 6,013,256; 5,693,761; and 6,180,370.

The rationale for using such primatized or humanized antibodies is toretain the (human) antigen specificity of the mouse antibody conferredby mouse CDRs but to reduce the immunogenicity of the mouse antibody (amouse antibody would cause an immune response against it in speciesother than the mouse) by using as much human framework sequence aspossible. Such antibodies may be used in human therapies for minimizingor eliminating unwanted side effects, such as immune responses.Antibodies comprising donor CDRs grafted from antigen-specific non-humanantibodies onto homologous non-human primate acceptor frameworks havingreduced immunogenicity in humans have been described (US 2005/0208625;US 2002/0062009; U.S. Pat. No. 7,338,658).

Accordingly, humanized forms of non-human (e.g. murine) antibodies arespecific chimeric immunoglobulins, immunoglobulin chains or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-bindingsubsequences of antibodies) that contain sequences derived fromnon-human immunoglobulin and human immunoglobulin sequences. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from the complementarity determining regions(CDRs) of the recipient antibody are replaced by residues from the CDRsof a non-human species (donor antibody) such as mouse, rat or rabbithaving the desired specificity, affinity and capacity. In someinstances, Fv framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human FR residues.

Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR or FRsequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody may comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRresidues are those of a human immunoglobulin consensus sequence. Thehumanized antibody optionally may also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

A humanized antibody (which includes, e.g., antibody fragments, andantibody derivatives or conjugates) may be produced by recombinant DNAtechnology, in which some or all of the amino acids of a humanimmunoglobulin light or heavy chain that are not required for antigenbinding (e.g., the constant regions and the framework regions of thevariable domains) are used to substitute for the corresponding aminoacids from the light or heavy chain of the cognate, non-human antibody.Methods for making humanized antibodies are well known to those of skillin the art of antibodies. See, e.g., EP 239400; Jones et al. (1986)Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536; Queen et al. (1989) Proc.Nat. Acad. Sci. USA 86:10029; Orlandi et al. (1989) Proc. Natl. Acad.Sci. USA 86:3833; U.S. Pat. No. 6,180,370; and EP 519596, whichdescribes antibody veneering of surface residues.

Accordingly, in one embodiment of this invention, an anti-CD154 antibodyrefers to a humanized antibody (which includes without limitation ahumanized antigen-binding fragment and a humanized antibody derivativeor a conjugate), that is generated by the transplantation of murine orrat (or other non-human) CDRs onto a human antibody. More specifically,this humanization is achieved as follows: (1) the cDNAs encoding heavyand light chain variable domains are isolated from a hybridoma or a Bcell that secretes the antibody; (2) the DNA sequences of the variabledomains, including the CDRs, are determined by sequencing; (3) the DNAsencoding the CDRs are transferred to the corresponding regions of ahuman antibody heavy or light chain variable domain coding sequence bysite directed mutagenesis; and (4) the human constant region genesegments of a desired isotype (e.g., 1 for CH and k for CL) are added.Finally, the humanized heavy and light chain genes are co-expressed inmammalian host cells (e.g., CHO or NS0 cells) to produce solublehumanized antibody.

At times, direct transfer of CDRs to a human framework leads to a lossof antigen-binding affinity of the resultant binding protein orantibody. Loss of antigen-binding affinity may occur because in somecognate antibodies, certain amino acids within the framework regionsinteract with the CDRs and thus influence the overall antigen bindingaffinity of the antibody. In such cases, the skilled worker willappreciate that it would be critical to introduce “back mutations” inthe framework regions of the acceptor antibody in order to retain theantigen-binding activity of the cognate antibody. The general approachesof making back mutations are well known to those of skill in the art.See, e.g., Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029; Co etal. 1991. Proc. Nat. Acad. Sci. USA 88:2869-2873; WO 90/07861; Tempest1991. Biotechnology 9: 266-271. Exemplary back mutations for antibodiesof the present invention include those residues depicted in FIG. 9 underdonor content.

In certain embodiments the binding protein or antibody of the presentinvention may comprise a V_(H) domain that is not a camelid or murineimmunoglobulin variable domain. In certain embodiments, the antibodypolypeptide may comprise a V_(H) domain that does not contain one ormore amino acids that are specific to camelid immunoglobulin variabledomains as compared to human V_(H) domains.

In one embodiment of this invention, an “anti-CD154 antibody” refers toan antibody (which includes an antigen-binding fragment and an antibodyderivative or conjugate) that is fully human. A fully “human” antibodycomprises an antibody polypeptide or an immunoglobulin variable domainthat has a sequence derived from a human immunoglobulin (e.g., obtainedfrom a human immunoglobulin coding sequence). The term “human antibody”includes, for example, antibodies having variable and constant regions(if present) derived from human germline immunoglobulin sequences. Theterm “human” as applied herein to an antibody or to a fragment such as avariable domain does not encompass an antibody from another species,e.g., mouse, that has been “humanized” through grafting of humanconstant region sequences onto an antibody polypeptide (i.e., replacingnon-human constant regions with human constant regions) or throughgrafting of human V region framework sequences onto an immunoglobulinvariable domain from a non-human mammal (i.e., replacing non-humanframework regions of a V domain with human framework regions). Methodsof humanizing immunoglobulin variable regions through rationalmodification of complementarity determining residues have been described(US 2006/0258852).

Human antibodies may, in certain embodiments, include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). In certain embodiments therefore, thepresent invention relates to an anti-CD154 antibody comprising avariable domain having one or more framework regions (e.g., FW1, FW2,FW3, and/or FW4) comprising an amino acid sequence that is the same asthe amino acid sequence of a corresponding framework region encoded by ahuman germline antibody gene segment, or the amino acid sequences of oneor more of said framework regions collectively comprising up to 5 aminoacid differences relative to the amino acid sequence of saidcorresponding framework region encoded by a human germline antibody genesegment. In further embodiments, the amino acid sequences of theframework regions (FW1, FW2, FW3, and FW4) of a variable domain are thesame as the amino acid sequences of corresponding framework regionsencoded by a human germline antibody gene segment, or the sequences ofFW1, FW2, FW3, and FW4 collectively contain up to 10 amino aciddifferences relative to the sequences of the corresponding frameworkregions encoded by the human germline antibody gene segment. Exemplarygermline antibody gene segments include, for example, DP47, DP45, DP48,and DPK9 (US 2006/0062784), and segments encoding the acceptor frameworksequences described in the Examples and Figures.

In some embodiments, a human antibody (which includes an antibodyfragment or variable domain sequence) has at least 85% amino acidsequence identity (including, for example, 87%, 90%, 93%, 95%, 97%, 99%or higher sequence identity) to a naturally-occurring human antibody.

Fully human antibodies may be derived from transgenic (e.g., geneticallyengineered such as knock-in) mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or human V, D and J regions from human cells. For example, it isnow possible to produce genetically engineered animals (e.g., mice) thatare capable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production (see,e.g., Jakobovits et al., PNAS, 90:2551 (1993); Jakobovits et al.,Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33(1993); and Duchosal et al., Nature, 355:258 (1992). A transgenic (e.g.,genetically engineered including knock-out, knock-in, gene replacementand the like) mouse strain may be engineered to contain gene sequencesfrom unrearranged human immunoglobulin genes. The human sequences maycode for both the heavy and light chains of human antibodies and wouldfunction correctly in the mice, undergoing rearrangement to provide awide antibody repertoire similar to that in humans. The geneticallyengineered mice may be immunized with the target protein (e.g., CD154,fragments thereof, or cells expressing CD154) to create a diverse arrayof specific antibodies and their encoding RNA. Nucleic acids encodingthe antibody chain components of such antibodies may then be cloned fromthe animal into a display vector. Typically, separate populations ofnucleic acids encoding heavy and light chain sequences are cloned, andthe separate populations then recombined on insertion into the vector,such that any given copy of the vector receives a random combination ofa heavy and a light chain. The vector is designed to express antibodychains so that they may be assembled and displayed on the outer surfaceof a display package containing the vector. For example, antibody chainsmay be expressed as fusion proteins with a phage coat protein from theouter surface of the phage. Thereafter, display packages may be screenedfor display of antibodies binding to a target.

In addition, human antibodies may be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996), U.S. Pat. No. 6,300,064). Synthetic phage libraries maybe created which use randomized combinations of synthetic human antibodyV-regions. Selection of antigen fully human antibodies may be made inwhich the V-regions are very human-like in nature. See U.S. Pat. Nos.6,794,132, 6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma2:361-367.

For the generation of human antibodies, also see Mendez et al. NatureGenetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med.188:483-495 (1998). Human antibodies are further discussed anddelineated in U.S. Pat. Nos. 5,939,598 and 6,673,986. Also see U.S. Pat.Nos. 6,114,598, 6,075,181, and 6,162,963. Also see U.S. Pat. Nos.6,150,584, 6,713,610, 6,657,103, US 2003/0229905 A1, US 2004/0010810 A1,US 2004/0093622 A1, US 2006/0040363 A1, US 2005/0054055 A1, US2005/0076395 A1 and US 2005/0287630 A1. See also EP 0463151 B1, WO94/02602, WO 96/34096, and WO 98/24893.

In an alternative approach, others have utilized a “minilocus” approach.In the minilocus approach, an exogenous Ig locus is mimicked through theinclusion of pieces (individual genes) from the Ig locus. Thus, one ormore VH genes, one or more DH genes, one or more JH genes, a mu constantregion, and a second constant region (preferably a gamma constantregion) are formed into a construct for insertion into an animal. Thisapproach is described in, e.g., U.S. Pat. Nos. 5,545,807, 5,545,806,5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650,5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and 5,643,763.Also see U.S. Pat. Nos. 5,569,825, 5,877,397, 6,300,129, 5,874,299,6,255,458, and 7,041,871, the disclosures of which are herebyincorporated by reference in their entirety. See also EP 0546073, WO92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884. Seefurther Taylor et al. (1992 Nuc. Acids Res., 20: 6287), Chen et al.(1993 Int. Immunol. 5: 647), Tuaillon et al. (1993 PNAS USA. 90:3720-4), Choi et al. (1993 Nature Genetics 4: 117), Lonberg et al. (1994Nature 368: 856-859), Taylor et al. (1994 International Immunology 6:579-591), Tuaillon et al. (1995 J Immunol. 154: 6453-65), Fishwild etal. (1996 Nature Biotechnology 14: 845), and Tuaillon et al. (2000 Eur JImmunol. 10: 2998-3005).

In a more particular embodiment of this invention, the fully humanantibodies are prepared using in vitro-primed human splenocytes (Boerneret al. 1991. J. Immunol. 147:86-95).

In a more particular embodiment of this invention, the fully humanantibodies are prepared by repertoire cloning (Persson et al. 1991.Proc. Nat. Acad. Sci. USA 88: 2432-2436; Huang and Stollar 1991. J.Immunol. Methods 141: 227-236). In addition, U.S. Pat. No. 5,798,230describes preparation of human monoclonal antibodies from human B cells,wherein human antibody-producing B cells are immortalized by infectionwith an Epstein-Barr virus, or a derivative thereof, that expressesEpstein-Barr virus nuclear antigen 2 (“EBNA2”), a protein required forimmortalization. The EBNA2 function is subsequently shut off, resultingin an increase in antibody production.

Other methods for producing fully human antibodies involve the use ofnon-human animals that have inactivated endogenous Ig loci and aretransgenic for un-rearranged human antibody heavy chain and light chaingenes. Such transgenic animals can be immunized with activated T cellsor the D1.1 protein (U.S. Pat. Nos. 5,474,771; 6,331,433; and 6,455,044)and hybridomas can be generated from B cells derived therefrom. Thedetails of these methods are described in the art. See, e.g. variouspublications/patents concerning transgenic mice containing human Igminiloci, including U.S. Pat. No. 5,789,650; the variouspublications/patents with respect to XENOMOUSE® (transgenic mice thatproduce full humanized antibodies), including U.S. Pat. Nos. 6,075,181;6,150,584; and 6,162,963; Green, 1997, Nature Genetics 7: 13-21; Mendez,1997, Nature Genetics 15: 146-56; and the various publications/patentsconcerning “transomic” mice, including EP 843961 and Tomizuka, 1997,Nature Genetics 16: 1433-43.

CD154 binding proteins and anti-CD154 antibodies of the presentinvention also relate to cross-blocking binding proteins or antibodies,or to binding proteins or antibodies that bind to the same epitope orthat bind to a closely related or overlapping epitope as any of theantibodies described herein. A cross-blocking binding protein orantibody can competitively inhibit or block binding of any of theantibodies described herein. In certain embodiments, the inventionprovides CD154 binding proteins and anti-CD154 antibodies that bind tothe same epitope as does a humanized antibody comprising a heavy chainsequence according to SEQ ID NO. 12 or SEQ ID NO. 13 and comprising alight chain sequence according to SEQ ID NO. 15 (342 Fab and Fab′fragments), and which exhibit similar CD154 binding properties incompetition assays with anti-CD154 antibody 5c8, as described herein. Inother embodiments, the invention provides CD154 binding proteins andanti-CD154 antibodies that bind to the same epitope as does a humanizedantibody comprising a V_(L) domain sequence according to SEQ ID NO. 58and a V_(H) domain sequence according to SEQ ID NO. 60 (338 antibodyvariable sequences), and which exhibit similar CD154 binding propertiesin competition assays with anti-CD154 antibody 5c8, as described herein.

In certain embodiments of the present invention, a CD154 bindingprotein, e.g. an anti-CD154 antibody, exhibits high affinity for humanCD154. For example in certain embodiments, a CD154 binding proteindissociates from human CD154 (human CD40L) with a K_(D) in the range of50 nM to 1 pM, inclusive, as determined by surface plasmon resonance(e.g., BIACORE® (an assay to measure molecular interactions)). Forexample, the K_(D) for human CD154 may be 25 nM to 1 pM, 10 nM to 1 pM,5 nm to 1 pM, 1 nM to 1 pM, 0.5 nM to 1 pM, 0.1 nM to 1 pM, 75 pM to 1pM, 50 pM to 1 pM, 20 pm to 1 pm, or even 10 pm to 1 pm. In otherembodiments, a CD154 binding protein of the present inventiondissociates from human CD154 with a K_(D) in the range of 500 pM to 1pM, inclusive, as determined by surface plasmon resonance (e.g.,BIACORE® (an assay to measure molecular interactions)). In someembodiments, the K_(D) for human CD154 is less than 50 pM. For example,in some of the embodiments of the present invention, a CD154 bindingprotein dissociates from human CD154 with a K_(D) that is less than 20pM. In some of the embodiments of the present invention, a CD154 bindingprotein dissociates from human CD154 with a K_(D) that is less than 10pM. In certain embodiments, a CD154 binding protein of the inventionbinds to CD154 with high affinity but does not displace bound CD154 fromCD40. Antibody affinity may be enhanced by methods known in the art(see, e.g., Clark et al. 2006 Protein Sci. 15(5): 949-60, whichdescribes affinity enhancement of an antibody using structure-basedcomputational design, and Chao et al. 2006 Nat Protoc 1: 755-768, whichdescribes methods of isolating and engineering scFvs with desiredproperties using yeast surface display).

Where it is desired to improve the affinity of antibodies of theinvention containing one or more of the above-mentioned CDRs, suchantibodies with improved affinity may be obtained by a number ofaffinity maturation protocols, including but not limited to maintainingthe CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chainshuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use ofmutation strains of E. coli. (Low et al., J. Mol. Biol., 250, 350-368,1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8,724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256,7-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998).All of these methods of affinity maturation are discussed by Vaughan etal. (Nature Biotechnology, 16, 535-539, 1998). Thus, the invention alsoprovides sequence variants of the antibodies of the invention which bindspecifically to CD154. Such sequence variants comprise one or moresemi-conservative or conservative substitutions within their sequenceand such substitutions preferably do not significantly affect thedesired activity of the polypeptide. Substitutions may be naturallyoccurring or may be introduced for example using mutagenesis (e.g.Hutchinson et al., 1978, J. Biol. Chem. 253:6551). The amino acidsglycine, alanine, valine, leucine and isoleucine, for example, can oftenbe substituted for one another (amino acids having aliphatic sidechains). Of these possible substitutions, it is preferred that glycineand alanine are used to substitute for one another (since they haverelatively short side chains) and that valine, leucine and isoleucineare used to substitute for one another (since they have larger aliphaticside chains which are hydrophobic). Other amino acids which may often besubstituted for one another include but are not limited to:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains).

The binding affinity of CD154 binding proteins, e.g., anti-CD154antibodies, of the present invention may also be described in relativeterms or as compared to the binding affinity of a second antibody thatalso specifically binds to CD154 (e.g., a second anti-CD154 antibodythat is CD154-specific, which may be referred to herein as a “secondCD154-specific antibody”). In some embodiments, the secondCD154-specific antibody may be antibody 5c8 (produced by the hybridomadeposited with ATCC under Accession No. HB 10916, as described in U.S.Pat. No. 5,474,771) or humanized 5c8. In other embodiments, the secondCD154-specific antibody may be any of the antibodies of the invention,such as 342, 381, 338, 294, 295, 300, 335, 303 or 402 (FIG. 12).Accordingly, certain embodiments of the present invention relate to ananti-CD154 antibody that binds to human CD154 with greater affinity thanantibody 5c8, or with a K_(D) that is lower than the K_(D) of antibody5c8. In certain embodiments, an anti-CD154 antibody of the presentinvention also inhibits CD154 activity or blocks the CD154:CD40interaction to a greater degree than does a second CD154-specificantibody, such as 5c8, at equimolar concentrations. The relative abilityof an anti-CD154 antibody to block the CD154:CD40 interaction may bemeasured by any available assay, such as the ICAM-1 upregulation assaydescribed herein and competition binding assays.

Accordingly, in certain embodiments, CD154 binding proteins andanti-CD154 antibodies (including antibody fragments and derivatives) ofthe invention are useful for inhibiting binding of CD154 to CD40 and doso with high specificity, e.g., with an IC50 in the range of 10 pM to1.5 μM, inclusive. In certain embodiments, the CD154 binding proteins,e.g., antibodies, of the invention may have an IC50 in the range of 10pm to 500 pm, 50 pm to 500 pm, 100 pm to 500 pm, 250 pm to 750 pm, 500pm to 1 μM or 750 pm to 1.5 μM. In further embodiments, the anti-CD154antibody does not substantially agonize CD40 activity or activate CD40signaling. In some embodiments, an anti-CD154 antibody of the presentinvention antagonizes an activity of CD154 or CD40 or both.

Certain embodiments of the present invention, therefore, relate to CD154binding proteins and anti-CD154 antibodies that bind to human CD154 withgreater or equal affinity relative to a second CD154-specific antibody(e.g., antibody 5c8 or humanized 5c8), or with a K_(D) that is lowerthan or equal to the K_(D) of a second CD154-specific antibody, whereinthe anti-CD154 antibody does not substantially inhibit the CD154:CD40interaction relative to the second CD154-specific antibody. Suchantibodies may be useful as binding assay and diagnostic reagents, forexample. Exemplary antibodies that exhibit a high affinity for humanCD154 but a lower degree of CD154:CD40 inhibition compared to a secondCD154-specific antibody are antibodies 381 and 338 described herein. Forexample, the present invention provides anti-CD154 antibodies thatspecifically bind to human CD154 with higher or equal affinity (relativeto a second CD154-specific antibody, such as humanized 5c8, for example)but that block the CD154:CD40 interaction to a lesser degree than doesthe second CD154-specific antibody. In further embodiments, theseanti-CD154 antibodies that inhibit binding of CD154 (CD40L) to CD40 to alesser degree than does a second CD154-specific antibody also do notsubstantially agonize CD40 activity or activate CD40 signaling. Forexample, such antibodies, if administered in an in vitro potency assayas described in Example 7, may not exhibit any statistically significantagonization over a control treatment or may exhibit 1%, 2%, 3%, 5%, or10% agonization compared to a positive control (e.g., CD154 ligand).Vidalain et al. (The EMBO Journal (2000) 19: 3304-3313) and Pearson etal. (International Immunology (2001) 13:273-283) describe the CD40signaling pathway and also provide assays that may be used to determinewhether or not the CD154:CD40 interaction is blocked or inhibited, andto what extent.

In some embodiments, the present invention relates to an anti-CD154antibody comprising at least one CDR selected from CDR-H1 (SEQ ID NO:3), CDR-H2 (SEQ ID NO: 4) and CDR-H3 (SEQ ID NO: 5). Preferably, theantibody comprises at least two CDRs selected from CDR-H1 (SEQ ID NO:3), CDR-H2 (SEQ ID NO: 4) and CDR-H3 (SEQ ID NO: 5) and more preferablyall three of CDR-H1, CDR-H2 and CDR-H3.

In another embodiment, the anti-CD154 antibody of the inventioncomprises at least one CDR selected from CDR-L1 (SEQ ID NO: 6), CDR-L2(SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8). Preferably, the antibodycomprises at least two CDRs selected from CDR-L 1 (SEQ ID NO: 6), CDR-L2(SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8) and more preferably all threeof CDR-L1, CDR-L2 and CDR-L3.

In further embodiments, the antibody comprises all three of CDR-H1,CDR-H2 and CDR-H3 (SEQ ID NOS: 3-5) and all three of CDR-L1, CDR-L2 andCDR-L3 (SEQ ID NOS: 6-8).

In further embodiments, the anti-CD154 antibody comprises a variableheavy chain sequence according to any one of SEQ ID NOS: 1, 9, 10 or 11and comprises a variable light chain sequence according to SEQ ID NO: 2or 14.

In some embodiments, the present invention relates to an anti-CD154antibody comprising at least one CDR selected from CDR-H1 (SEQ ID NO:42), CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQ ID NO: 44). Preferably, theantibody comprises at least two CDRs selected from CDR-H1 (SEQ ID NO:42), CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQ ID NO: 44) and morepreferably all three of CDR-H1, CDR-H2 and CDR-H3.

In another embodiment, the anti-CD154 antibody of the inventioncomprises at least one CDR selected from CDR-L1 (SEQ ID NO: 45), CDR-L2(SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47). Preferably, the antibodycomprises at least two CDRs selected from CDR-L 1 (SEQ ID NO: 45),CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47) and more preferablyall three of CDR-L1, CDR-L2 and CDR-L3.

In further embodiments, the antibody comprises all three of CDR-H1,CDR-H2 and CDR-H3 (SEQ ID NOS: 42-44) and all three of CDR-L1, CDR-L2and CDR-L3 (SEQ ID NOS: 45-47).

In further embodiments, the anti-CD154 antibody comprises a variableheavy chain sequence according to SEQ ID NO: 56 and/or comprises avariable light chain sequence according to SEQ ID NO: 54.

In some embodiments, the present invention relates to an anti-CD154antibody comprising at least one CDR selected from CDR-H1 (SEQ ID NO:48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50). Preferably, theantibody comprises at least two CDRs selected from CDR-H1 (SEQ ID NO:48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50) and morepreferably all three of CDR-H1, CDR-H2 and CDR-H3.

In another embodiment, the anti-CD154 antibody of the inventioncomprises at least one CDR selected from CDR-L1 (SEQ ID NO: 51), CDR-L2(SEQ ID NO: 52) and CDR-L3 (SEQ ID NO: 53). Preferably, the antibodycomprises at least two CDRs selected from CDR-L1 (SEQ ID NO: 51), CDR-L2(SEQ ID NO: 52) and CDR-L3 (SEQ ID NO: 53) and more preferably all threeof CDR-L1, CDR-L2 and CDR-L3.

In certain embodiments, the antibodies have a complementary sequencecomprising one or more light chain CDRs of CDR-L1, CDR-L2 and CDR-L3,above, or a complementary sequence comprising one or more heavy chainCDRs of CDR-H1, CDR-H2 and CDR-H3, above, respectively. Thus, in certainembodiments, an antibody of this invention comprises CDR-H1 (SEQ ID NO:48), CDR-H2 (SEQ ID NO: 49) or CDR-H3 (SEQ ID NO: 50), and CDR-L1 (SEQID NO: 51), CDR-L2 (SEQ ID NO: 52) or CDR-L3 (SEQ ID NO: 53).

In further embodiments, the antibody comprises all three of CDR-H1,CDR-H2 and CDR-H3 (SEQ ID NOS: 48-50) and all three of CDR-L1, CDR-L2and CDR-L3 (SEQ ID NOS: 51-53).

In further embodiments, the anti-CD154 antibody comprises a variableheavy chain sequence according to SEQ ID NO: 60 and/or comprises avariable light chain sequence according to SEQ ID NO: 58.

In certain embodiments, the CD154 binding protein of this inventioncomprises a light chain sequence according to SEQ ID NO: 62 and a heavychain sequence according to SEQ ID NO: 65. In other embodiments, theCD154 binding protein of this invention comprises a light chain sequenceaccording to SEQ ID NO: 63 and a heavy chain sequence according to SEQID NO: 66.

In certain embodiments, the CD154 binding protein of this inventioncomprises a light chain sequence according to SEQ ID NO: 68 and a heavychain sequence according to SEQ ID NO: 71. In other embodiments, theCD154 binding protein of this invention comprises a light chain sequenceaccording to SEQ ID NO: 69 and a heavy chain sequence according to SEQID NO: 72. In other embodiments, the CD154 binding protein comprises oneof the sequences according to SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:71 or SEQ ID NO: 72.

This invention also provides CD154 binding proteins that preferablyshare at least 90%, 91%, 92%, 93% or 94% identity with a CD154 bindingprotein of the invention. More preferably, a CD154 binding proteinshares at least 95%, 96%, 97% or 98% identity. Most preferably, a CD154binding protein shares at least 99%, 99.5%, 99.9% or more identity witha CD154 binding protein of the invention. The CD154 binding proteins ofthe invention may comprise a variable domain sequence is at least 85%identical to a variable domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 or SEQ ID NO: 60, orone or more CDRs thereof.

Other embodiments of the invention relate to bispecific antibodies.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. They may be used alone or mixed into compositions comprisingpolyclonal populations. In the present invention, one of the bindingspecificities is for the CD154 antigen while the other bindingspecificity is for any other antigen, and preferably for a cell-surfaceprotein or receptor or receptor subunit. For example, the other bindingspecificity may be a ligand selected from among human serum albumin(HSA), TNFα, IL-1, IL-2, IL-4, IL-6, IL-8, IL-12, IL-18, IFN-γ, CD2,CD4, CD8, CTLA4, LFA1, LFA3 and VLA4.

In other embodiments of the present invention, an anti-CD154 antibodycomprises a generic ligand binding site. A generic ligand (e.g., apolypeptide) is capable of binding functional members of a repertoireregardless of target ligand specificity. A generic ligand may thereforebe used to identify or select (for example, as in purification andscreening processes) functional members of a repertoire (such as acollection or group of antibodies, regardless of the antibodies' antigenbinding specificities). Generic ligands include, for example, Protein A,Protein G and Protein L. Pre-selection of members of a phage librarywith generic ligands is taught in WO 99/20749.

Antibody Fragments

The present invention also relates to antigen-binding or epitope-bindinganti-CD154 antibody fragments. All of the methods and reagents describedabove with respect to anti-CD154 antibodies may similarly be used toproduce and use anti-CD154 antibody fragments of this invention.

In some embodiments of this invention, the anti-CD154 antibody fragmentsinclude heteromeric antibody complexes and antibody fusions, such asbispecific antibodies, hemidimeric antibodies, multivalent antibodies(i.e., tetravalent antibodies) and single-chain antibodies. Ahemidimeric antibody is made up of an Fc portion and one Fab portion. Asingle chain antibody is made up of variable regions linked by proteinspacers in a single protein chain.

In some embodiments of this invention, the anti-CD154 antibody fragmentsof this invention may also include proteins containing one or moreimmunoglobulin light chains and/or heavy chains, such as monomers andhomo- or hetero-multimers (e.g., dimers or trimers) of these chains,where these chains are optionally disulfide-bonded or otherwisecross-linked. These antibodies may be capable of binding to one or moreantigens.

In certain embodiments, the present invention includes antigen-bindingfragments of whole antibodies, such as Fab, F(ab)₂, Fab′, F(ab′)₂,F(ab′)₃, F(v), Fd, dAb, diabody, minibody, and nanobody antibodyfragments. The present invention also relates to fragments comprisingonly a single variable domain, such as a V_(H) or V_(L) domain, or to afragment comprising only the heavy chain or light chain domains. Thefragments may be humanized or fully human. The present invention alsoincludes antigen-binding fragments that are made from alternativescaffolds (see, e.g., Binz supra and Hosse supra) or that comprise auniversal framework. The present invention also relates to conjugatescomprising any antigen-binding fragment or CD154 binding protein thatspecifically binds CD154 conjugated covalently or noncovalently, ordirectly or indirectly, to a functional moiety such as a carrier proteinor PEG, for example.

Anti-CD154 antibodies of the present invention include divalentantibodies. Thus, in certain embodiments, the invention relates to adivalent anti-CD154 antibody fragment comprising two antibody heavychains and at least one polymer molecule in covalent linkage, each heavychain being covalently linked to the other by at least one non-disulfideinterchain bridge linking the sulfur atom of a cysteine residue in onechain to the sulfur atom of a cysteine residue in the other chain, thecysteine residues being located outside of the variable region domain ofeach chain, characterized in that at least one non-disulfide interchainbridge contains a covalently linked polymer molecule. The term“non-disulfide” as used herein is intended to mean that S—S bridges,e.g. of the type normally found in antibodies, are excluded. Aninterchain bridge of the type present in a fragment according to theinvention may however still be linked to a heavy chain via an S—S-bondas described hereinafter. In general, each polymer molecule in thedivalent antibody fragment according to the invention forms part of aninterchain bridge. Each bridge serves to link two heavy chains and eachchain will be covalently linked to a sulphur atom of a cysteine residue.The covalent linkage will generally be a disulfide bond or, inparticular embodiments, a sulfur-carbon bond. For exemplary divalentantibody structures, see WO 99/64460 and WO 05/061005.

The invention also provides an anti-CD154 antibody that is a monovalentantibody or monovalent antigen-binding fragment. As used herein, theterm “monovalent” means that a given antibody or antigen-bindingfragment (e.g., Fv, a single chain scFv, dAb, Fab, Fab′, Fd, scFab,scFabΔC, etc.) can bind only a single molecule of its target.Naturally-occurring antibodies are generally divalent, in that they havetwo functional antigen-binding arms, each comprising a V_(H) and a V_(L)domain. A divalent antibody can bind two separate molecules of the sameantigen where steric hindrance is not an issue. In contrast, a“monovalent” antibody has one antigen-binding site for a target. Theantigen-binding domain of a monovalent antibody may comprise a V_(H) anda V_(L) domain or may comprise only a single immunoglobulin variabledomain, i.e., a V_(H) or a V_(L) domain, that has the capacity to bindCD154 without the need for a corresponding V_(L) or V_(H) domain,respectively. Such an exemplary monovalent antibody is an Fd fragmentthat comprises a single immunoglobulin variable domain and that can onlybind to one CD154 antigen molecule. A monovalent antibody lacks thecapacity to cross link molecules of a single antigen.

This invention provides an anti-CD154 antibody, wherein the antibodyspecifically binds to an epitope to which a humanized Fab or Fab′fragment with a heavy chain sequence according to SEQ ID NO: 12 or 13,respectively and with a light chain sequence according to SEQ ID NO: 15specifically binds. In certain embodiments, an antibody of thisinvention is an antibody fragment with a variable heavy chain sequenceaccording to SEQ ID NO: 1, 9, 10 or 11 and with a variable light chainsequence according to SEQ ID NO: 2 or 14. In other embodiments, theantibody of this invention is an antibody fragment with a heavy chainsequence according to SEQ ID NO. 12 or 13 and a light chain sequenceaccording to SEQ ID NO. 15.

In certain embodiments, the CD154 binding protein or antibody comprisesor consists of a light chain sequence of SEQ ID NO: 15 and a heavy chainsequence of SEQ ID NO: 13. In certain embodiments, the CD154 bindingprotein or antibody comprises or consists of a light chain sequence ofSEQ ID NO: 15 and a heavy chain sequence of SEQ ID NO: 12.

In alternative embodiments, the invention provides an antibody fragmentcomprising a variable heavy chain sequence according to SEQ ID NO: 56and comprising a variable light chain sequence according to SEQ ID NO:54. In other embodiments, the antibody of this invention is an antibodyfragment comprising a variable heavy chain sequence according to SEQ IDNO. 60 and comprising a variable light chain sequence according to SEQID NO. 58.

In one embodiment, this invention provides a Fab′ or a F(ab′)₂ or aF(ab′)₃ fragment that specifically binds CD154. This Fab′ or F(ab′)₂ orF(ab′)₃ fragment may be humanized and may have a heavy chain sequencethat comprises or consists of the sequence of SEQ ID NO: 13 and may havea light chain sequence that comprises or consists of the sequence of SEQID NO: 15.

In some embodiments, the present invention relates to an anti-CD154antibody that is free of an Fc domain. Such an Fc-deficient antibody orfragment may be monovalent, divalent, or further multivalent.

This invention also provides antibodies or antibody fragments that bindspecifically to the epitope to which the Fab′ or F(ab′)₂ or F(ab′)₃fragments described above specifically bind. These antibodies orantibody fragments may be identified by a cross-blocking assay asdescribed herein (Example 9). These antibodies or antibody fragments maybe isolated, recombinant or synthetic and may be attached to a secondmolecule to form an antibody-conjugate.

Antibodies with Reduced Effector Function

The interaction of antibodies and antibody-antigen complexes with cellsof the immune system triggers a variety of responses, referred to hereinas effector functions. IgG antibodies activate effector pathways of theimmune system by binding to members of the family of cell surface Fcγreceptors and to C1q of the complement system. Ligation of effectorproteins by clustered antibodies triggers a variety of responses,including release of inflammatory cytokines, regulation of antigenproduction, endocytosis, and cell killing. In some clinical applicationsthese responses are crucial for the efficacy of a monoclonal antibody.In others they provoke unwanted side effects such as inflammation andthe elimination of antigen-bearing cells. Accordingly, the presentinvention further relates to CD154 binding proteins, includingantibodies, with altered, e.g., reduced, effector functions.Importantly, reduced effector function does not necessarily reduce theability of an anti-CD154 antibody to inhibit one or several diseases viablocking the CD154-CD40 interaction (see WO 05/03175, which isincorporated herein by reference in its entirety).

Anti-CD154 antibodies with diminished effector function (e.g.,Fc-mediated effector functions; see below) are particularly desirablefor use in subjects where the potential for undesirable thromboembolicactivity exists. Additionally, the diminished effector function ofanti-CD154 antibodies may decrease or eliminate other potentialundesired side effects of anti-CD154 antibody therapies, such asdeletion of activated T cells and other populations of cells induced toexpress CD154 or Fc-dependent activation of monocytes/macrophages.

Effector function of an anti-CD154 antibody of the present invention maybe determined using one of many known assays. The anti-CD154 antibody'seffector function may be reduced relative to a second anti-CD154antibody. In some embodiments, the second anti-CD154 antibody may be anyantibody that binds CD154 specifically. In some embodiments, the secondanti-CD154 antibody may be antibody 5c8 (produced by the hybridomadeposited with ATCC under Accession No. HB 10916, as described in U.S.Pat. No. 5,474,771) or humanized 5c8. In other embodiments, the secondCD154-specific antibody may be any of the antibodies of the invention,such as 342, 381, 338, 294, 295, 300, 335, 303 or 402 (FIG. 12). Inother embodiments, where the anti-CD154 antibody of interest has beenmodified to reduce effector function, the second anti-CD154 antibody maybe the unmodified or parental version of the antibody.

Effector function of an anti-CD154 antibody of the present invention mayalso be determined, e.g., by measuring the level of platelet aggregationor activation caused by treatment with the anti-CD154 antibody relativeto a control antibody. In some embodiments, therefore, the anti-CD154antibodies of the present invention do not mediate or do not enhanceplatelet aggregation or activation in a standard platelet aggregation oractivation assay. In other embodiments, the anti-CD154 antibodiesmediate a lower level of platelet aggregation or activation relative toa second anti-CD154 antibody (e.g., 5c8 or humanized 5c8).

Exemplary effector functions include Fc receptor binding, phagocytosis,apoptosis, pro-inflammatory responses, down-regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc. Other effector functionsinclude antibody-dependent cell-mediated cytotoxicity (ADCC), wherebyantibodies bind Fc receptors on cytotoxic T cells, natural killer (NK)cells, or macrophages leading to cell death, and complement-dependentcytotoxicity (CDC), which is cell death induced via activation of thecomplement cascade (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234(1997); Ward and Ghetie, Therapeutic Immunol. 2:77-94 (1995); andRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)). Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g. an antibody variable domain) and can be assessed usingstandard assays that are known in the art (see, e.g., WO 05/018572, WO05/003175, and U.S. Pat. No. 6,242,195).

Effector functions can be avoided by using antibody fragments lackingthe Fc domain such as Fab, Fab′2, or single chain Fv. An alternative hasbeen to use the IgG4 subtype antibody, which binds to FcγRI but whichbinds poorly to C1q and FcγRII and RIII The IgG2 subtype also hasreduced binding to Fc receptors, but retains significant binding to theH131 allotype of FcγRIIa and to C1q. Thus, additional changes in the Fcsequence are required to eliminate binding to all the Fc receptors andto C1q.

Several antibody effector functions, including ADCC, are mediated by Fcreceptors (FcRs), which bind the Fc region of an antibody. The affinityof an antibody for a particular FcR, and hence the effector activitymediated by the antibody, may be modulated by altering the amino acidsequence and/or post-translational modifications of the Fc and/orconstant region of the antibody.

FcRs are defined by their specificity for immunoglobulin isotypes; Fcreceptors for IgG antibodies are referred to as FcγR, for IgE as FcεR,for IgA as FcαR and so on. Three subclasses of FcγR have beenidentified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Both FcγRIIand FcγRIII have two types: FcγRIIA (CD32) and FcγRIIB (CD32); andFcγRIIIA (CD16a) and FcγRIIIB (CD16b). Because each FcγR subclass isencoded by two or three genes, and alternative RNA splicing leads tomultiple transcripts, a broad diversity in FcγR isoforms exists. Forexample, FcγRII (CD32) includes the isoforms 11a, 11b1, 11b2, 11b3, and11c.

The binding site on human and murine antibodies for FcγR has beenpreviously mapped to the so-called “lower hinge region” consisting ofresidues 233-239 (EU index numbering as in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), Woof et al. Molec.Immunol. 23:319-330 (1986); Duncan et al. Nature 332:563 (1988);Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel etal., Proc. Natl. Acad. Sci USA 88:9036-9040 (1991)). Of residues233-239, P238 and S239 are among those cited as possibly being involvedin binding. Other previously cited areas possibly involved in binding toFcγR are: G316-K338 (human IgG) for human FcγRI (by sequence comparisononly; no substitution mutants were evaluated) (Woof et al. MolecImmunol. 23:319-330 (1986)); K274-R301 (human IgG1) for human FcγRIII(based on peptides) (Sarmay et al. Molec. Immunol. 21:43-51 (1984)); andY407-R416 (human IgG) for human FcγRIII (based on peptides) (Gergely etal. Biochem. Soc. Trans. 12:739-743 (1984), Shields et al. J Biol Chem276: 6591-6604 (2001), Lazar G A et al. Proc Natl Acad Sci 103:4005-4010 (2006)). These and other stretches or regions of amino acidresidues involved in FcR binding may be evident to the skilled artisanfrom an examination of the crystal structures of Ig-FcR complexes (see,e.g., Sondermann et al. 2000 Nature 406(6793):267-73 and Sondermann etal. 2002 Biochem Soc Trans. 30(4):481-6). Accordingly, the anti-CD154antibodies of the present invention include modifications of one or moreof the aforementioned residues.

Other known approaches for reducing mAb effector function includemutating amino acids on the surface of the mAb that are involved ineffector binding interactions (Lund, J., et al. (1991) J. Immunol.147(8): 2657-62; Shields, R. L. et al. (2001) J. Biol. Chem. 276(9):6591-604; and using combinations of different subtype sequence segments(e.g., IgG2 and IgG4 combinations) to give a greater reduction inbinding to Fcγ receptors than either subtype alone (Armour et al., Eur.J. Immunol. (1999) 29: 2613-2624; Mol. Immunol. 40 (2003) 585-593).

A large number of Fc variants having altered and/or reduced affinitiesfor some or all Fc receptor subtypes (and thus for effector functions)are known in the art. See, e.g., US 2007/0224188; US 2007/0148171; US2007/0048300; US 2007/0041966; US 2007/0009523; US 2007/0036799; US2006/0275283; US 2006/0235208; US 2006/0193856; US 2006/0160996; US2006/0134105; US 2006/0024298; US 2005/0244403; US 2005/0233382; US2005/0215768; US 2005/0118174; US 2005/0054832; US 2004/0228856; US2004/132101; US 2003/158389; see also U.S. Pat. Nos. 7,183,387;6,737,056; 6,538,124; 6,528,624; 6,194,551; 5,624,821; 5,648,260.

In CDC, the antibody-antigen complex binds complement, resulting in theactivation of the complement cascade and generation of the membraneattack complex. Activation of the classical complement pathway isinitiated by the binding of the first component of the complement system(C1q) to antibodies (of the appropriate subclass) which are bound totheir cognate antigen; thus the activation of the complement cascade isregulated in part by the binding affinity of the immunoglobulin to C1qprotein. To activate the complement cascade, it is necessary for C1q tobind to at least two molecules of IgG1, IgG2, or IgG3, but only onemolecule of IgM, attached to the antigenic target (Ward and Ghetie,Therapeutic Immunology 2:77-94 (1995) p. 80). To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., JImmunol. Methods 202:163 (1996) may be performed.

It has been proposed that various residues of the IgG molecule areinvolved in binding to C1q including the Glu318, Lys320 and Lys322residues on the CH2 domain, amino acid residue 331 located on a turn inclose proximity to the same beta strand, the Lys235 and Gly237 residueslocated in the lower hinge region, and residues 231 to 238 located inthe N-terminal region of the CH2 domain (see, e.g., Xu et al., JImmunol. 150:152A (Abstract) (1993); WO94/29351; Tao et al., J. Exp.Med., 178:661-667 (1993); Brekke et al., Eur. J. Immunol., 24:2542-47(1994); Burton et al., Nature, 288:338-344 (1980); Duncan and Winter,Nature 332:738-40 (1988); Idusogie et al., J Immunol 164: 4178-4184(2000); U.S. Pat. Nos. 5,648,260; and 5,624,821). As an example in IgG1,two mutations in the COOH terminal region of the CH2 domain of humanIgG1—K322A and P329A—do not activate the CDC pathway and were shown toresult in more than a 100 fold decrease in C1q binding (U.S. Pat. No.6,242,195).

Thus, in certain embodiments of the invention, one or more of theseresidues may be modified, substituted, or removed or one or more aminoacid residues may be inserted so as to decrease CDC activity of theCD154 antibodies provided herein. For example in some embodiments, itmay be desirable to reduce or eliminate effector function(s) of thesubject antibodies in order to reduce or eliminate the potential offurther activating immune responses. Antibodies with decreased effectorfunction may also reduce the risk of thromboembolic events in subjectsreceiving the antibodies.

In certain other embodiments, the present invention provides ananti-CD154 antibody that exhibits reduced binding to one or more FcRreceptors but that maintains its ability to bind complement (e.g., to asimilar or, in some embodiments, to a lesser extent than a native,non-variant, or parent anti-CD154 antibody). Accordingly, an anti-CD154antibody of the present invention may bind and activate complement whileexhibiting reduced binding to an FcR, such as FcγRIIa (e.g., FcγRIIaexpressed on platelets). Such an antibody with reduced or no binding toFcγRIIa (such as FcγRIIa expressed on platelets) but that can bind C1qand activate the complement cascade to at least some degree will reducethe risk of thromboembolic events while maintaining perhaps desirableeffector functions. In alternative embodiments, an anti-CD154 antibodyof the present invention exhibits reduced binding to one or more FcRsbut maintains its ability to bind one or more other FcRs. See, forexample, US 2007-0009523, 2006-0194290, 2005-0233382, 2004-0228856, and2004-0191244, which describe various amino acid modifications thatgenerate antibodies with reduced binding to FcRI, FcRII, and/or FcRIII,as well as amino acid substitutions that result in increased binding toone FcR but decreased binding to another FcR.

Accordingly, effector functions involving the constant region of ananti-CD154 antibody may be modulated by altering properties of theconstant region, and the Fc region in particular. In certainembodiments, the anti-CD154 antibody having reduced effector function iscompared with a second antibody with effector function and which may bea non-variant, native, or parent antibody (e.g., antibody 342 orantibody 5c8, which is described in U.S. Pat. No. 5,474,771) comprisinga native constant or Fc region that mediates effector function. Inparticular embodiments, effector function modulation includes situationsin which an activity is abolished or completely absent.

A native sequence Fc or constant region comprises an amino acid sequenceidentical to the amino acid sequence of an Fc or constant chain regionfound in nature. Preferably, a control molecule used to assess relativeeffector function comprises the same type/subtype Fc region as does thetest or variant antibody. A variant or altered Fc or constant regioncomprises an amino acid sequence which differs from that of a nativesequence heavy chain region by virtue of at least one amino acidmodification (such as post-translational modification, amino acidsubstitution, insertion, or deletion). Accordingly, the variant constantregion may contain one or more amino acid substitutions, deletions, orinsertions that result in altered post-translational modifications,including, for example, an altered glycosylation pattern. A parentantibody or Fc region is, for example, a variant having normal effectorfunction used to construct a constant region (i.e., Fc) having altered,e.g., reduced, effector function.

Antibodies with altered (e.g., reduced or eliminated) effectorfunction(s) may be generated by engineering or producing antibodies withvariant constant, Fc, or heavy chain regions. Recombinant DNA technologyand/or cell culture and expression conditions may be used to produceantibodies with altered function and/or activity. For example,recombinant DNA technology may be used to engineer one or more aminoacid substitutions, deletions, or insertions in regions (such as Fc orconstant regions) that affect antibody function including effectorfunctions. Alternatively, changes in post-translational modifications,such as glycosylation patterns (see below), may be achieved bymanipulating the host cell and cell culture and expression conditions bywhich the antibody is produced.

Amino acid alterations, such as amino acid substitutions, can alter theeffector function of the anti-CD154 antibodies of the present inventionwithout affecting antigen binding affinity. The amino acid substitutionsdescribed above (e.g., Glu318, Kys320, Lys332, Lys235, Gly237, K332, andP329), for example, may be used to generate antibodies with reducedeffector function.

In other embodiments, amino acid substitutions may be made for one ormore of the following amino acid residues: 234, 235, 236, 237, 297, 318,320, and 322 of the heavy chain constant region (see U.S. Pat. Nos.5,624,821 and 5,648,260). Such substitutions may alter effector functionwhile retaining antigen binding activity. An alteration at one or moreof amino acids 234, 235, 236, and 237 can decrease the binding affinityof the Fc region for FcγRI receptor as compared to an unmodified ornon-variant antibody. Amino acid residues 234, 236, and/or 237 may besubstituted with alanine, for example, and amino acid residue 235 may besubstituted with glutamine, for example. In another embodiment, ananti-CD154 IgG1 antibody may comprise a substitution of Leu at position234 with Ala, a substitution of Leu at position 235 with Glu, and asubstitution of Gly at position 237 with Ala.

Additionally or alternatively, the Fc amino acid residues at 318, 320,and 322 may be altered. These amino acid residues, which are highlyconserved in mouse and human IgGs, mediate complement binding. It hasbeen shown that alteration of these amino acid residues reduces C1qbinding but does not alter antigen binding, protein A binding, or theability of the Fc to bind to mouse macrophages.

In another embodiment, an anti-CD154 antibody of the present inventionis an IgG4 immunoglobulin comprising substitutions that reduce oreliminate effector function. The IgG4 Fc portion of an anti-CD154antibody of the invention may comprise one or more of the followingsubstitutions: substitution of proline for glutamate at residue 233,alanine or valine for phenylalanine at residue 234 and alanine orglutamate for leucine at residue 235 (EU numbering, Kabat, E. A. et al.(1991), supra). Further, removing the N-linked glycosylation site in theIgG4 Fc region by substituting Ala for Asn at residue 297 (EU numbering)may further reduce effector function and eliminate any residual effectoractivity that may exist. Another exemplary IgG4 mutant with reducedeffector function is the IgG4 subtype variant containing the mutationsS228P and L235E (PE mutation) in the heavy chain constant region. Thismutation results in reduced effector function. See U.S. Pat. Nos.5,624,821 and 5,648,260. Another exemplary mutation in the IgG4 contextthat reduces effector function is S228P/T229A, as described herein.

Other exemplary amino acid sequence changes in the constant regioninclude but are not limited to the Ala-Ala mutation described byBluestone et al. (see WO 94/28027 and WO 98/47531; also see Xu et al.2000 Cell Immunol 200: 16-26). Thus in certain embodiments, anti-CD154antibodies with mutations within the constant region including theAla-Ala mutation may be used to reduce or abolish effector function.According to these embodiments, the constant region of an anti-CD154antibody comprises a mutation to an alanine at position 234 or amutation to an alanine at position 235. Additionally, the constantregion may contain a double mutation: a mutation to an alanine atposition 234 and a second mutation to an alanine at position 235.

In one embodiment, an anti-CD154 antibody comprises an IgG4 framework,wherein the Ala-Ala mutation would describe a mutation(s) fromphenylalanine to alanine at position 234 and/or a mutation from leucineto alanine at position 235. In another embodiment, the anti-CD154antibody comprises an IgG1 framework, wherein the Ala-Ala mutation woulddescribe a mutation(s) from leucine to alanine at position 234 and/or amutation from leucine to alanine at position 235. An anti-CD154 antibodymay alternatively or additionally carry other mutations, including thepoint mutation K322A in the CH2 domain (Hezareh et al. 2001 J. Virol.75: 12161-8).

Other exemplary amino acid substitutions are provided in WO 94/29351(which is incorporated herein by reference in its entirety), whichrecites antibodies having mutations in the N-terminal region of the CH2domain that alter the ability of the antibodies to bind to FcRI, therebydecreasing the ability of antibodies to bind to C1q which in turndecreases the ability of the antibodies to fix complement. Also see Coleet al. (J. Immunol. (1997) 159: 3613-3621), which describes mutations inthe upper CH2 regions in IgG2 that result in lower FcR binding.

Methods of generating any of the aforementioned antibody variantscomprising amino acid substitutions are well known in the art. Thesemethods include, but are not limited to, preparation by site-directed(or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of a prepared DNA molecule encoding the antibody or at leastthe constant region of the antibody.

Site-directed mutagenesis is well known in the art (see, e.g., Carter etal. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel et al., Proc.Natl. Acad. Sci. USA 82:488 (1987)).

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183(Academic Press, 1990) and Vallette et al., Nuc. Acids Res. 17:723-733(1989).

Another method for preparing sequence variants, cassette mutagenesis, isbased on the technique described by Wells et al., Gene 34:315-323(1985).

Another embodiment of the present invention relates to an anti-CD154antibody with reduced effector function in which the antibody's Fcregion, or portions thereof, is swapped with an Fc region (or withportions thereof) having naturally reduced effector inducing activity.For example, human IgG4 constant region exhibits reduced or nocomplement activation. Further, the different IgG molecules differ intheir binding affinity for FcR, which may be due at least in part to thevarying length and flexibility of the IgGs' hinge regions (whichdecreases in the order IgG3>IgG1>IgG4>IgG2). For example, IgG4 exhibitsreduced or no binding to FcγRIIa. For examples of chimeric molecules andchimeric constant regions, see, e.g., Gillies et al. (Cancer Res. 1999,59: 2159-2166) and Mueller et al. (Mol. Immunol. 1997, 34: 441-452).

The invention also relates to anti-CD154 antibodies with reducedeffector function in which the Fc region is completely absent. Suchantibodies may also be referred to as antibody derivatives andantigen-binding fragments of the present invention. Such derivatives andfragments may be fused to non-antibody protein sequences or non-proteinstructures, especially structures designed to facilitate delivery and/orbioavailability when administered to an animal, e.g., a human subject(see below).

As discussed above, changes within the hinge region also affect effectorfunctions.

For example, deletion of the hinge region may reduce affinity for Fcreceptors and may reduce complement activation (Klein et al. 1981 PNASUSA 78: 524-528). The present disclosure therefore also relates toantibodies with alterations in the hinge region.

In particular embodiments, antibodies of the present invention may bemodified to inhibit complement dependent cytotoxicity (CDC). ModulatedCDC activity may be achieved by introducing one or more amino acidsubstitutions, insertions, or deletions in an Fc region of the antibody(see, e.g., U.S. Pat. Nos. 6,194,551 and 6,242,195). Alternatively oradditionally, cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved or reducedinternalization capability and/or increased or decreasedcomplement-mediated cell killing. See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992), WO99/51642, Duncan & Winter Nature 322: 738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351.

It is further understood that effector function may vary according tothe binding affinity of the antibody. For example, antibodies with highaffinity may be more efficient in activating the complement systemcompared to antibodies with relatively lower affinity (Marzocchi-Machadoet al. 1999 Immunol Invest 28: 89-101). Accordingly, an antibody may bealtered such that the binding affinity for its antigen is reduced (e.g.,by changing the variable regions of the antibody by methods such assubstitution, addition, or deletion of one or more amino acid residues).An antibody with reduced binding affinity may exhibit reduced effectorfunctions, including, for example, reduced ADCC and/or CDC.

Anti-CD154 antibodies of the present invention with reduced effectorfunction include antibodies with reduced binding affinity for one ormore Fc receptors (FcRs) relative to a parent or non-variant anti-CD154antibody. Accordingly, anti-CD154 antibodies with reduced FcR bindingaffinity include anti-CD154 antibodies that exhibit a 1.5-fold, 2-fold,2.5-fold, 3-fold, 4-fold, or 5-fold or higher decrease in bindingaffinity to one or more Fc receptors compared to a parent or non-variantanti-CD154 antibody (e.g., antibody 342 or 5c8). In some embodiments, ananti-CD154 antibody with reduced effector function binds to an FcR withabout 10-fold less affinity relative to a parent or non-variantantibody. In other embodiments, an anti-CD154 antibody with reducedeffector function binds to an FcR with about 15-fold less affinity orwith about 20-fold less affinity relative to a parent or non-variantantibody. The FcR receptor may be one or more of FcγRI (CD64), FcγRII(CD32), and FcγRIII, and isoforms thereof, and FcεR, FcμR, FcδR, and/orFcαR. In particular embodiments, an anti-CD154 antibody with reducedeffector function exhibits a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold,or 5-fold or higher decrease in binding affinity to FcγRIIa.

Accordingly, in certain embodiments, an anti-CD154 antibody of thepresent invention exhibits reduced binding to a complement proteinrelative to a second anti-CD154 antibody. In certain embodiments, ananti-CD154 antibody of the invention exhibits reduced binding by afactor of about 1.5-fold or more, about 2-fold or more, about 3-fold ormore, about 4-fold or more, about 5-fold or more, about 6-fold or more,about 7-fold or more, about 8-fold or more, about 9-fold or more, about10-fold or more, or about 15-fold or more relative to a secondanti-CD154 antibody.

Accordingly, in certain embodiments, the present invention relates toantibodies that elicit reduced effector function when administered to asubject. In certain embodiments, an anti-CD154 antibody of thisinvention does not cause thrombosis in a subject to whom the antibody isadministered. Thrombosis includes, for example, thromboembolic events.Such events include, e.g., vasculopathy (e.g., vascular changes such asintimal thickening and vessel wall changes). In some embodiments, ananti-CD154 antibody of this invention causes fewer thromboembolic eventsrelative to a second CD154-specific antibody (e.g., antibody 342,antibody 5c8 or humanized 5c8). An anti-CD154 antibody with reducedeffector function may show a 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%reduction in the number of thromboembolic events when administered to asubject and compared to a non-variant or parent anti-CD154 antibody.

In certain embodiments, an anti-CD154 antibody of this invention doesnot cause platelet aggregation or activation in vitro and/or enhancesplatelet aggregation or activation to a lesser extent when compared to asecond CD154-specific antibody (e.g., antibody 5c8 or humanized 5c8; seeExample 1). Accordingly, in certain embodiments, the presence of a CD154binding protein, e.g., an anti-CD154 antibody, of the present inventionin a standard platelet aggregation or activation assay does not resultin aggregation or activation of more than 20%, 25%, 30% or 50% over theaggregation or activation observed in a negative control assay. Anexemplary standard platelet aggregation assay is the assay describedherein (Example 11, Assay 1) and shown in FIG. 26. Both a standard assayand an alternative platelet aggregation assay (Assay 2, FIG. 27) thatmay be used in the invention are described in Example 11. The CD154binding protein may be soluble.

In certain embodiments, this invention also provides CD154 bindingprotein, e.g., an anti-CD154 antibody, said protein comprising a singleimmunoglobulin variable domain which specifically and monovalently bindsCD40L, wherein binding of said soluble protein to CD154 does notsubstantially induce JNK phosphorylation in Jurkat T-cells. Thisinvention also provides CD154 binding protein, e.g., an anti-CD154antibody, said protein comprising a single immunoglobulin variabledomain which specifically and monovalently binds CD154, wherein bindingof said soluble protein to CD154 does not substantially induce IFN-gammasecretion by Jurkat T-cells co-stimulated with anti-CD3 antibody.

Certain embodiments of the present invention relate to an anti-CD154antibody comprising one or more heavy chain CDR sequences selected fromCDR-H1 (SEQ ID NO: 3), CDR-H2 (SEQ ID NO: 4) and CDR-H3 (SEQ ID NO: 5),wherein the antibody further comprises a variant Fc region that confersreduced effector function compared to a native or parental Fc region. Infurther embodiments, the anti-CD154 antibody comprises at least two ofthe CDRs, and in other embodiments the antibody comprises all threeheavy chain CDR sequences, which are CDR-H1 (SEQ ID NO: 3), CDR-H2 (SEQID NO: 4) and CDR-H3 (SEQ ID NO: 5).

Other embodiments of the present invention relate to an anti-CD154antibody comprising one or more light chain CDR sequences selected fromCDR-L1 (SEQ ID NO: 6), CDR-L2 (SEQ ID NO: 7) and CDR-L3 (SEQ ID NO: 8),the antibody further comprising a variant Fc region that confers reducedeffector function compared to a native or parental Fc region. In furtherembodiments, the anti-CD154 antibody comprises at least two of the lightchain CDRs, and in other embodiments the antibody comprises all threelight chain CDR sequences, which are CDR-L1 (SEQ ID NO: 6), CDR-L2 (SEQID NO: 7) and CDR-L3 (SEQ ID NO: 8).

In further embodiments of the present invention, the anti-CD154 antibodywith reduced effector function comprises all three light chain CDRsequences, which are CDR-L1 (SEQ ID NO: 6), CDR-L2 (SEQ ID NO: 7) andCDR-L3 (SEQ ID NO: 8), and comprises all three heavy chain CDRsequences, which are CDR-H1 (SEQ ID NO: 3), CDR-H2 (SEQ ID NO: 4) andCDR-H3 (SEQ ID NO: 5).

In certain embodiments, this invention provides an anti-CD154 antibodythat specifically binds a CD154 protein, wherein the antibody comprisesa V_(H) sequence selected from SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10and SEQ ID NO: 11. In certain other embodiments, this invention providesan antibody that specifically binds a CD154 protein, wherein theantibody comprises a heavy chain sequence selected from SEQ ID NO: 12and SEQ ID NO: 13. In further embodiments, the anti-CD154 antibodycomprising any one or more of the CDRs or heavy or light chain sequencesdescribed above is a Fab or a Fab′ fragment or a derivative thereof. Inyet further embodiments, the antibody is a F(ab′)₂ fragment or aderivative thereof. Other antibody fragments or derivatives thereofcomprising the CDRs or the heavy or light chain sequences thatspecifically bind a CD154 protein are also included. These antibodiesmay be modified so as to elicit reduced or no effector functions.Accordingly, in certain embodiments, the present invention relates to ananti-CD154 antibody comprising a V_(H) sequence selected from SEQ ID NO:1, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, the antibody furthercomprising a variant Fc region that confers reduced effector functioncompared to a native or parental Fc region.

In other embodiments, the invention relates to an anti-CD154 antibodycomprising a V_(L) sequence selected from SEQ ID NO: 2 and SEQ ID NO:14, the antibody further comprising a variant Fc region that confersreduced effector function compared to a native or parental Fc region.

In some embodiments, an antibody comprises a variable light chainsequence of SEQ ID NO: 2. In further embodiments, an antibody comprisingSEQ ID NO: 2 is a Fab or a Fab′ fragment or a derivative thereof. In yetother embodiments, the antibody is a F(ab′)₂ fragment or a derivativethereof. Other antibody fragments or derivatives thereof comprising theCDRs or the heavy or light chain sequences that specifically bind aCD154 protein are also included.

In additional embodiments, the invention relates to an anti-CD154antibody comprising a V_(L) sequence selected from the group consistingof SEQ ID NO: 2 and SEQ ID NO: 14 and further comprising a V_(H)sequence selected from SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10 and SEQID NO: 11, the antibody further comprising a variant Fc region thatconfers reduced effector function compared to a native or parental Fcregion.

In other embodiments, the invention relates to an anti-CD154 antibodycomprising a V_(H) sequence of SEQ ID NO: 29 and a V_(L) sequence of SEQID NO: 30, the antibody further comprising a variant Fc region thatconfers reduced effector function compared to a native or parental Fcregion.

In certain embodiments, the present invention provides an anti-CD154antibody that specifically binds a CD154 protein, wherein the antibodycomprises the light chain sequence of SEQ ID NO: 15. In furtherembodiments, the antibody comprising the light chain sequence of SEQ IDNO: 15 further comprises a variant Fc region that confers reducedeffector function compared to a native or parental Fc region (e.g., anFc region with altered glycosylation and/or other modification).

In certain embodiments, the present invention relates to an anti-CD154antibody comprising a light chain sequence of SEQ ID NO: 15 and a heavychain sequence selected from the group consisting of SEQ ID NO: 12 andSEQ ID NO: 13, the antibody further comprising a variant Fc region thatconfers reduced effector function compared to a native or parental Fcregion (e.g., an Fc region with altered glycosylation and/or othermodification).

In certain embodiments, the invention provides an anti-CD154 antibodycomprising a light chain sequence as set forth in SEQ ID NO: 2 or 14 anda heavy chain sequence as set forth in SEQ ID NO: 1, 9, 10 or 11. Insome embodiments, the anti-CD154 antibody may be an antibody fragment.In further embodiments, the antibody is a Fab or a Fab′ fragment or aderivative thereof. In yet further embodiments, the antibody is aF(ab′)₂ fragment or a derivative thereof. Other antibody fragments orderivatives thereof comprising the CDRs or the heavy or light chainsequences that specifically bind a CD154 protein are also included.Additionally, the antibodies may exhibit reduced effector function. Forexample, antibodies comprising an Fc region may comprise an Fc regionwith one or more modifications (e.g., altered glycosylation,conjugation, etc.) such that the antibody elicits reduced or no effectorfunction(s).

In other embodiments, the present invention relates to an anti-CD154antibody comprising a light chain sequence of SEQ ID NO: 15 and a heavychain sequence of SEQ ID NO: 13, wherein the antibody further comprisesa variant Fc region that confers reduced effector function compared to anative or parental Fc region. In other embodiments, the presentinvention relates to an anti-CD154 antibody comprising a light chainsequence of SEQ ID NO: 15 and a heavy chain sequence of SEQ ID NO: 12,wherein the antibody further comprises a variant Fc region that confersreduced effector function compared to a native or parental Fc region.

In some embodiments, the present invention relates to an anti-CD154antibody comprising one or more heavy chain CDR sequences selected fromCDR-H1 (SEQ ID NO: 42), CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQ ID NO:44), the antibody further comprising a variant Fc region that confersreduced effector function compared to a native or parental Fc region. Incertain embodiments, the anti-CD154 antibody comprises at least two ofthe heavy chain CDRs, and in other embodiments the antibody comprisesall three heavy chain CDR sequences, which are CDR-H1 (SEQ ID NO: 42),CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQ ID NO: 44).

In certain embodiments, the invention relates to an anti-CD154 antibodycomprising one or more light chain CDR sequences selected from CDR-L1(SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47), theantibody further comprising a variant Fc region that confers reducedeffector function compared to a native or parental Fc region. In certainembodiments, the anti-CD154 antibody comprises at least two of the lightchain CDRs, and in other embodiments the antibody comprises all threelight chain CDR sequences, which are CDR-L1 (SEQ ID NO: 45), CDR-L2 (SEQID NO: 46) and CDR-L3 (SEQ ID NO: 47).

Further embodiments of the present invention relate to an anti-CD154antibody comprising a variant Fc region that confers reduced effectorfunction compared to a native or parental Fc region, wherein theantibody comprises all three light chain CDR sequences, which are CDR-L1(SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47), andwherein the antibody comprises all three heavy chain CDR sequences,which are CDR-H1 (SEQ ID NO: 42), CDR-H2 (SEQ ID NO: 43) and CDR-H3 (SEQID NO: 44).

In other embodiments, the present invention relates to an anti-CD154antibody comprising one or more heavy chain CDR sequences selected fromCDR-H1 (SEQ ID NO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO:50), the antibody further comprising a variant Fc region that confersreduced effector function compared to a native or parental Fc region. Infurther embodiments, the anti-CD154 antibody comprises at least two ofthe heavy chain CDRs, and in other embodiments the antibody comprisesall three heavy chain CDR sequences, which are CDR-H1 (SEQ ID NO: 48),CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50).

In certain embodiments, the present invention relates to an anti-CD154antibody comprising one or more light chain CDR sequences selected fromCDR-L1 (SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3 (SEQ ID NO:53), the antibody further comprising a variant Fc region that confersreduced effector function compared to a native or parental Fc region. Infurther embodiments, the antibody comprises at least two of the lightchain CDRs, and in other embodiments the antibody comprises all threelight chain CDR sequences, which are CDR-L1 (SEQ ID NO: 51), CDR-L2 (SEQID NO: 52) and CDR-L3 (SEQ ID NO: 53).

In certain embodiments, an anti-CD154 antibody with reduced effectorfunction comprises all three light chain CDR sequences, which are CDR-L1(SEQ ID NO: 51), CDR-L2 (SEQ ID NO: 52) and CDR-L3 (SEQ ID NO: 53), andfurther comprises all three heavy chain CDR sequences, which are CDR-H1(SEQ ID NO: 48), CDR-H2 (SEQ ID NO: 49) and CDR-H3 (SEQ ID NO: 50).

In further embodiments, this invention provides an anti-CD154 antibodythat specifically binds CD154, wherein the antibody comprises a V_(L)sequence of SEQ ID NO: 54. The invention also relates to an anti-CD154antibody that comprises a V_(H) sequence of SEQ ID NO: 56. An anti-CD154antibody of the invention may comprise both a V_(L) sequence of SEQ IDNO: 54 and a V_(H) sequence of SEQ ID NO: 56. In further embodiments,the antibody is a Fab or a Fab′ fragment or a derivative thereof. In yetfurther embodiments, the antibody or antibody fragment is a F(ab′)₂fragment or a derivative thereof. The antibody or fragment may also be ahumanized or fully human antibody or fragment thereof. Certainembodiments of the present invention relate to an anti-CD154 antibodycomprising a V_(H) sequence of SEQ ID NO: 56 and V_(L) sequence of SEQID NO: 54, the antibody further comprising a variant Fc region thatconfers reduced effector function compared to a native or parental Fcregion.

In further embodiments, this invention provides an anti-CD154 antibodythat specifically binds CD154, wherein the antibody comprises a V_(L)sequence of SEQ ID NO: 58. In additional embodiments, the antibodycomprises a V_(H) sequence of SEQ ID NO: 60. In further embodiments, theantibody or fragment comprises a V_(H) sequence of SEQ ID NO: 60 and aV_(L) sequence of SEQ ID NO: 58. The antibody may be a Fab or a Fab′fragment or a derivative thereof. In yet further embodiments, theantibody is a F(ab′)₂ fragment or a derivative thereof. The antibody mayalso be a humanized or fully human antibody or fragment thereof. Inother embodiments, the present invention relates to an anti-CD154antibody comprising a V_(H) sequence of SEQ ID NO: 58 and V_(L) sequenceof SEQ ID NO: 60, the antibody further comprising a variant Fc regionthat confers reduced effector function compared to a native or parentalFc region.

Anti-CD154 Antibodies with Altered Glycosylation

Glycan removal produces a structural change that should greatly reducebinding to all members of the Fc receptor family across species. Inglycosylated antibodies, including anti-CD154 antibodies, the glycans(oligosaccharides) attached to the conserved N-linked site in the CH2domains of the Fc dimer are enclosed between the CH2 domains, with thesugar residues making contact with specific amino acid residues on theopposing CH2 domain. Different glycosylation patterns are associatedwith different biological properties of antibodies (Jefferis and Lund,1997, Chem. Immunol., 65: 111-128; Wright and Morrison, 1997, TrendsBiotechnol., 15: 26-32). Certain specific glycoforms confer potentiallyadvantageous biological properties. Loss of the glycans changes spacingbetween the domains and increases their mobility relative to each otherand is expected to have an inhibitory effect on the binding of allmembers of the Fc receptor family. For example, in vitro studies withvarious glycosylated antibodies have demonstrated that removal of theCH2 glycans alters the Fc structure such that antibody binding to Fcreceptors and the complement protein C1Q are greatly reduced. Anotherknown approach to reducing effector functions is to inhibit productionof or remove the N-linked glycans at position 297 (EU numbering) in theCH2 domain of the Fc (Nose et al., 1983 PNAS 80: 6632; Leatherbarrow etal., 1985 Mol. Immunol. 22: 407; Tao et al., 1989 J. Immunol. 143: 2595;Lund et al., 1990 Mol. Immunol. 27: 1145; Dorai et al., 1991 Hybridoma10:211; Hand et al., 1992 Cancer Immunol. Immunother. 35:165; Leader etal., 1991 Immunology 72: 481; Pound et al., 1993 Mol. Immunol. 30:233;Boyd et al., 1995 Mol. Immunol. 32: 1311). It is also known thatdifferent glycoforms can profoundly affect the properties of atherapeutic, including pharmacokinetics, pharmacodynamics,receptor-interaction and tissue-specific targeting (Graddis et al.,2002, Curr Pharm Biotechnol. 3: 285-297). In particular, for antibodies,the oligosaccharide structure can affect properties relevant to proteaseresistance, the serum half-life of the antibody mediated by the FcRnreceptor, phagocytosis and antibody feedback, in addition to effectorfunctions of the antibody (e.g., binding to the complement complex C1,which induces CDC, and binding to FcγR receptors, which are responsiblefor modulating the ADCC pathway) (Nose and Wigzell, 1983; Leatherbarrowand Dwek, 1983; Leatherbarrow et al., 1985; Walker et al., 1989; Carteret al., 1992, PNAS, 89: 4285-4289).

Accordingly, another means of modulating effector function of antibodiesincludes altering glycosylation of the antibody constant region. Alteredglycosylation includes, for example, a decrease or increase in thenumber of glycosylated residues, a change in the pattern or location ofglycosylated residues, as well as a change in sugar structure(s). Theoligosaccharides found on human IgGs affect their degree of effectorfunction (Raju, T. S., BioProcess International, April 2003, 44-53); themicroheterogeneity of human IgG oligosaccharides can affect biologicalfunctions such as CDC and ADCC, binding to various Fc receptors, andbinding to C1q protein (Wright A. & Morrison S. L., TIBTECH 1997, 1526-32; Shields et al. J Biol Chem. 2001 276(9):6591-604; Shields et al.J Blot Chem. 2002 277(30):26733-40; Shinkawa et al. J Blot Chem. 2003278(5):3466-73; Umana et al. Nat Biotechnol. 1999 February, 17(2):176-80). For example, the ability of IgG to bind C1q and activate thecomplement cascade may depend on the presence, absence or modificationof the carbohydrate moiety positioned between the two CH2 domains (whichis normally anchored at Asn297) (Ward and Ghetie, Therapeutic Immunology2:77-94 (1995)).

Glycosylation sites in an Fc-containing polypeptide, for example anantibody such as an IgG antibody, may be identified by standardtechniques. The identification of the glycosylation site can beexperimental or based on sequence analysis or modeling data. Consensusmotifs, that is, the amino acid sequence recognized by various glycosyltransferases, have been described. For example, the consensus motif foran N-linked glycosylation motif is frequently NXT or NXS, where X can beany amino acid except proline. Several algorithms for locating apotential glycosylation motif have also been described. Accordingly, toidentify potential glycosylation sites within an antibody orFc-containing fragment, the sequence of the antibody is examined, forexample, by using publicly available databases such as the websiteprovided by the Center for Biological Sequence Analysis (see NetNGlycservices for predicting N-linked glycosylation sites and NetOGlycservices for predicting O-linked glycosylation sites).

In vivo studies have confirmed the reduction in the effector function ofaglycosyl antibodies. For example, an aglycosyl anti-CD8 antibody isincapable of depleting CD8-bearing cells in mice (Isaacs, 1992 J.Immunol. 148: 3062) and an aglycosyl anti-CD3 antibody does not inducecytokine release syndrome in mice or humans (Boyd, 1995 supra; Friend,1999 Transplantation 68:1632).

Importantly, while removal of the glycans in the CH2 domain appears tohave a significant effect on effector function, other functional andphysical properties of the antibody remain unaltered. Specifically, ithas been shown that removal of the glycans had little to no effect onserum half-life and binding to antigen (Nose, 1983 supra; Tao, 1989supra; Dorai, 1991 supra; Hand, 1992 supra; Hobbs, 1992 Mol. Immunol.29:949).

Although there is in vivo validation of the aglycosyl approach, thereare reports of residual effector function with aglycosyl mAbs (see,e.g., Pound, J. D. et al. (1993) Mol. Immunol. 30(3): 233-41; Dorai, H.et al. (1991) Hybridoma 10(2): 211-7). Armour et al. show residualbinding to FcγRIIa and FcγRIIb proteins (Eur. J. Immunol. (1999) 29:2613-1624; Mol. Immunol. 40 (2003) 585-593). Thus a further decrease ineffector function, particularly complement activation, may be importantto guarantee complete ablation of activity in some instances. For thatreason, aglycosyl forms of IgG2 and IgG4 and a G1/G4 hybrid areenvisioned as being useful in methods and antibody compositions of theinvention having reduced effector functions.

Generation of Deglycosylated or Aglycosyl Anti-CD154 Antibodies

The anti-CD154 antibodies of the present invention may be modified oraltered to elicit reduced effector function(s) (compared to a secondCD154-specific antibody) while optionally retaining the other valuableattributes of the Fc portion.

Accordingly, in certain embodiments, the present invention relates toaglycosyl anti-CD154 antibodies with decreased effector function, whichare characterized by a modification at the conserved N-linked site inthe CH2 domains of the Fc portion of the antibody. A modification of theconserved N-linked site in the CH2 domains of the Fc dimer can lead toaglycosyl anti-CD154 antibodies. Examples of such modifications includemutation of the conserved N-linked site in the CH2 domains of the Fcdimer, removal of glycans attached to the N-linked site in the CH2domains, and prevention of glycosylation. For example, an aglycosylanti-CD154 antibody may be created by changing the canonical N-linkedAsn site in the heavy chain CH2 domain to a Gln residue (see, forexample, WO 05/03175 and US 2006-0193856).

In one embodiment of present invention, the modification comprises amutation at the heavy chain glycosylation site to prevent glycosylationat the site. Thus, in one embodiment of this invention, the aglycosylanti-CD154 antibodies are prepared by mutation of the heavy chainglycosylation site, i.e., mutation of N298Q (N297 using Kabat EUnumbering) and expressed in an appropriate host cell. For example, thismutation may be accomplished by following the manufacturer's recommendedprotocol for unique site mutagenesis kit from AMERSHAM-PHARMACIABIOTECH®, a biotechnology company (Piscataway, N.J., USA).

The mutated antibody can be stably expressed in a host cell (e. g. NSOor CHO cell) and then purified. As one example, purification can becarried out using Protein A and gel filtration chromatography. It willbe apparent to those of skill in the art that additional methods ofexpression and purification may also be used.

In another embodiment of the present invention, the aglycosyl anti-CD154antibodies have decreased effector function, wherein the modification atthe conserved N-linked site in the CH2 domains of the Fc portion of saidantibody or antibody derivative comprises the removal of the CH2 domainglycans, i.e., deglycosylation. These aglycosyl anti-CD154 antibodiesmay be generated by conventional methods and then deglycosylatedenzymatically. Methods for enzymatic deglycosylation of antibodies arewell known to those of skill in the art (Williams, 1973; Winkelhake &Nicolson, 1976 J Biol Chem. 251:1074-80.).

In another embodiment of this invention, deglycosylation may be achievedby growing host cells which produce the antibodies in culture mediumcomprising a glycosylation inhibitor such as tunicamycin (Nose &Wigzell, 1983). That is, the modification is the reduction or preventionof glycosylation at the conserved N-linked site in the CH2 domains ofthe Fc portion of said antibody.

In other embodiments of this invention, recombinant CD154 polypeptides(or cells or cell membranes containing such polypeptides) may be used asan antigen to generate an anti-CD154 antibody or antibody derivatives,which may then be deglycosylated.

In alternative embodiments, agyclosyl anti-CD154 antibodies oranti-CD154 antibodies with reduced glycosylation of the presentinvention may be produced by the method described in Taylor et al. (WO05/18572 and US 2007-0048300). For example, in one embodiment, ananti-CD154 aglycosyl antibody may be produced by altering a first aminoacid residue (e.g., by substitution, insertion, deletion, or by chemicalmodification), wherein the altered first amino acid residue inhibits theglycosylation of a second residue by either steric hindrance or chargeor both. In certain embodiments, the first amino acid residue ismodified by amino acid substitution. In further embodiments, the aminoacid substitution is selected from the group consisting of Gly, Ala,Val, Leu, Ile, Phe, Asn, Gln, Trp, Pro, Ser, Thr, Tyr, Cys, Met, Asp,Glu, Lys, Arg, and His. In other embodiments, the amino acidsubstitution is a non-traditional amino acid residue. The second aminoacid residue may be near or within a glycosylation motif, for example,an N-linked glycosylation motif that contains the amino acid sequenceNXT or NXS. In one exemplary embodiment, the first amino acid residue isamino acid 299 and the second amino acid residue is amino acid 297,according to the Kabat numbering. For example, the first amino acidsubstitution may be T299A, T299N, T299G, T299Y, T299C, T299H, T299E,T299D, T299K, T299R, T299G, T299I, T299L, T299M, T299F, T299P, T299W, orT299V, according to the Kabat numbering. In particular embodiments, theamino acid substitution is T299C.

Effector function may also be reduced by modifying an antibody of thepresent invention such that the antibody contains a blocking moiety.Exemplary blocking moieties include moieties of sufficient steric bulkand/or charge such that reduced glycosylation occurs, for example, byblocking the ability of a glycosidase to glycosylate the polypeptide.The blocking moiety may additionally or alternatively reduce effectorfunction, for example, by inhibiting the ability of the Fc region tobind a receptor or complement protein. In some embodiments, the presentinvention relates to a CD154 binding protein, e.g., an anti-CD154antibody, comprising a heavy chain CDR3 sequence selected from SEQ IDNOS: 5, 44 and 50, and a variant Fc region, the variant Fc regioncomprising a first amino acid residue and an N-glycosylation site, thefirst amino acid residue modified with side chain chemistry to achieveincreased steric bulk or increased electrostatic charge compared to theunmodified first amino acid residue, thereby reducing the level of orotherwise altering glycosylation at the N-glycosylation site. In certainof these embodiments, the variant Fc region confers reduced effectorfunction compared to a control, non-variant Fc region. In furtherembodiments, the side chain with increased steric bulk is a side chainof an amino acid residue selected from the group consisting of Phe, Trp,His, Glu, Gln, Arg, Lys, Met and Tyr. In yet further embodiments, theside chain chemistry with increased electrostatic charge is a side chainof an amino acid residue selected from the group consisting of Asp, Glu,Lys, Arg, and His.

Accordingly, in one embodiment, glycosylation and Fc binding can bemodulated by substituting T299 with a charged side chain chemistry suchas D, E, K, or R. The resulting antibody will have reduced glycosylationas well as reduced Fc binding affinity to an Fc receptor due tounfavorable electrostatic interactions.

In another embodiment, a T299C variant antibody, which is bothaglycosylated and capable of forming a cysteine adduct, may exhibit lesseffector function (e.g., FcγRI binding) compared to its aglycosylatedantibody counterpart (see, e.g., WO 05/18572). Accordingly, alterationof a first amino acid proximal to a glycosylation motif can inhibit theglycosylation of the antibody at a second amino acid residue; when thefirst amino acid is a cysteine residue, the antibody may exhibit evenfurther reduced effector function. In addition, inhibition ofglycosylation of an antibody of the IgG4 subtype may have a moreprofound affect on FcγRI binding compared to the effects ofaglycosylation in the other subtypes.

In additional embodiments, the present invention relates to anti-CD154antibodies with altered glycosylation that exhibit reduced binding toone or more FcR receptors and that optionally also exhibit increased ornormal binding to one or more Fc receptors and/or complement—e.g.,antibodies with altered glycosylation that at least maintain the same orsimilar binding affinity to one or more Fc receptors and/or complementas a native, control anti-CD154 antibody. For example, anti-CD154antibodies with predominantly Man₅G1cNAc₂N-glycan as the glycanstructure present (e.g., wherein Man₅G1cNAc₂N-glycan structure ispresent at a level that is at least about 5 percent more than the nextpredominant glycan structure of the Ig composition) may exhibit alteredeffector function compared to an anti-CD154 antibody population whereinMan₅G1cNAc₂N-glycan structure is not predominant. Antibodies withpredominantly this glycan structure exhibit decreased binding to FcγRIIaand FcγRIIb, increased binding to FcγRIIIa and FcγRIIIb, and increasedbinding to C1q subunit of the C1 complex (see US 2006-0257399). Thisglycan structure, when it is the predominant glycan structure, confersincreased ADCC, increased CDC, increased serum half-life, increasedantibody production of B cells, and decreased phagocytosis bymacrophages.

In general, the glycosylation structures on a glycoprotein will varydepending upon the expression host and culturing conditions (Raju, T.S., BioProcess International April 2003, 44-53). Such differences canlead to changes in both effector function and pharmacokinetics (Israelet al. Immunology, 1996, 89(4):573-578; Newkirk et al. P. Clin. Exp.1996, 106(2):259-64). For example, galactosylation can vary with cellculture conditions, which may render some immunoglobulin compositionsimmunogenic depending on their specific galactose pattern (Patel et al.,1992, Biochem J. 285: 839-845). The oligosaccharide structures ofglycoproteins produced by non-human mammalian cells tend to be moreclosely related to those of human glycoproteins. Further, proteinexpression host systems may be engineered or selected to express apredominant Ig glycoform or alternatively may naturally produceglycoproteins having predominant glycan structures. Examples ofengineered protein expression host systems producing a glycoproteinhaving a predominant glycoform include gene knockouts/mutations (Shieldset al., 2002, JBC, 277: 26733-26740); genetic engineering (Umana et al.,1999, Nature Biotech., 17: 176-180) or a combination of both.Alternatively, certain cells naturally express a predominantglycoform—for example, chickens, humans and cows (Raju et al., 2000,Glycobiology, 10: 477-486). Thus, the expression of an anti-CD154antibody or antibody composition having altered glycosylation (e.g.,predominantly one specific glycan structure) can be obtained by oneskilled in the art by selecting at least one of many expression hostsystems. Protein expression host systems that may be used to produceanti-CD154 antibodies of the present invention include animal, plant,insect, bacterial cells and the like. For example, US 2007-0065909,2007-0020725, and 2005-0170464 describe producing aglycosylatedimmunoglobulin molecules in bacterial cells. As a further example,Wright and Morrison produced antibodies in a CHO cell line deficient inglycosylation (1994 J Exp Med 180: 1087-1096) and showed that antibodiesproduced in this cell line were incapable of complement-mediatedcytolysis. Other examples of expression host systems found in the artfor production of glycoproteins include: CHO cells: Raju WO 99/22764 andPresta WO 03/35835; hybridoma cells: Trebak et al., 1999, J. Immunol.Methods, 230: 59-70; insect cells: Hsu et al., 1997, JBC, 272:9062-9070;and plant cells: Gerngross et al., WO 04/74499. To the extent that agiven cell or extract has resulted in the glycosylation of a givenmotif, art recognized techniques for determining if the motif has beenglycosylated are available, for example, using gel electrophoresisand/or mass spectroscopy.

Additional methods for altering glycosylation sites of antibodies aredescribed, e.g., in U.S. Pat. Nos. 6,350,861 and 5,714,350, WO 05/18572and WO 05/03175; these methods can be used to produce anti-CD154antibodies of the present invention with altered, reduced, or noglycosylation.

The aglycosyl anti-CD154 antibodies with reduced effector function maybe antibodies that comprise modifications or that may be conjugated tocomprise a functional moiety. Such moieties include a blocking moiety(e.g., a PEG moiety, cysteine adducts, etc.), a detectable moiety (e.g.,fluorescent moieties, radioisotopic moieties, radiopaque moieties, etc.,including diagnostic moieties), and/or a therapeutic moiety (e.g.,cytotoxic agents, anti-inflammatory agents, immunomodulatory agents,anti-infective agents, anti-cancer agents, anti-neurodegenerativeagents, radionuclides, etc.).

Antibody Conjugates

When administered, antibodies are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of antibodies may be required to sustain thetherapeutic efficacy of the antibody treatment.

In one embodiment of this invention, the anti-CD154 antibodies of thisinvention may be antibodies modified (e.g., attached to other moietiessuch as a heterologous functional moiety) to increase the integrity andlongevity of the antibody in vivo. For example, the anti-CD154antibodies of this invention may be antibodies that are modified toinclude a moiety (functional moiety) that can increase stabilization,thereby prolonging the serum half-life of the antibody. The serum halflife of a CD154 binding protein, e.g., antibody, of the invention may beat least 3 days, at least 7 days, at least 14 days, at least 21 days, atleast 28 days, at least 1 month or more. Such functional moieties thatincrease the half-life of the antibodies may be particularly useful inembodiments where the antibody is an antibody fragment, for example.

Antibody modifications may also increase the protein's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the protein, and greatly reduce the immunogenicityand antigenicity of the protein. As a result, the desired in vivobiological activity may be achieved by the administration of suchpolymer-protein adducts less frequently or in lower doses than with theunmodified protein.

Accordingly, in certain embodiments, the antibodies of the invention areantibodies attached to heterologous functional moieties to form antibodyconjugates. A “functional moiety” refers to any functional moiety (e.g.,polypeptide, protein domain, carrier, polymer, etc.) that is associatedwith an antibody of the invention. Association with an anti-CD154antibody may be by covalent or non-covalent attachment and may also bereversible or regulatable. Exemplary molecules that may be used to formCD154 antibody conjugates of the present invention include but are notlimited to functional moieties such as antineoplastic agents, drugs,toxins, biologically active proteins, for example enzymes, otherantibodies, antibody derivatives or antibody fragments, synthetic (forexample, PEG) or naturally occurring polymers, nucleic acids andfragments thereof, e.g. DNA, RNA and fragments thereof, aptamers,radionuclides, particularly radioiodide, radioisotopes, chelated metals,nanoparticles and reporter groups such as fluorescent or luminescentcompounds or compounds which may be detected by imaging techniques suchas NMR or ESR spectroscopy.

In a further example, a functional moiety to which the antibodies of theinvention are conjugated may increase the half-life of the antibody invivo, and/or reduce immunogenicity of the antibody and/or enhance thedelivery of an antibody across an epithelial barrier to the immunesystem. Examples of suitable functional moieties of this type includepolymers, dextran, hydroxypropylmethacrylamide (HPMA), transferrin,albumin, albumin binding proteins or albumin binding compounds such asthose described in PCT/GB2005/002084.

Where the functional moiety is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide. See for example Veronese and Pasut,2005, Drug Discovery Today, 10(21):1451-1458; Pasut et al., 2004, ExpertOpinion in Therapeutic Patents, 14(6): 859-894.

Particular optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups.

Particular examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinyl alcohol) or derivatives thereof, especially optionallysubstituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) orderivatives thereof.

Particular naturally occurring polymers include lactose, amylose,dextran, glycogen or derivatives thereof “Derivatives” in this contextis intended to include reactive derivatives, for example thiol-selectivereactive groups such as maleimides and the like. The reactive group maybe linked directly or through a linker segment to the polymer. It willbe appreciated that the residue of such a group will in some instancesform part of the product as the linking group between the antibodyfragment and the polymer.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50,000 Da,preferably from 5000 Da to 40,000 Da and more preferably from 20,000 Dato 40,000 Da. The polymer size may in particular be selected on thebasis of the intended use of the product, for example ability tolocalize to certain tissues such as tumors or extend circulatinghalf-life (for a review see Chapman, 2002, Advanced Drug DeliveryReviews, 54, 531-545). Thus, for example, where the product is intendedto leave the circulation and penetrate tissue, for example for use inthe treatment of a tumor, it may be advantageous to use a smallmolecular weight polymer, for example with a molecular weight of around5000 Da. For applications where the product remains in the circulation,it may be advantageous to use a higher molecular weight polymer, forexample having a molecular weight in the range from 20,000 Da to 40,000Da.

Particularly preferred polymers include a polyalkylene polymer, such asa poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) ora derivative thereof, and especially with a molecular weight in therange from about 15,000 Da to about 40,000 Da.

Preferably, the antibody, antibody derivative or antibody fragment ofthis invention, including an antibody or fragment with reduced effectorfunction, is attached to a polyalkylene polymer, particularly apoly(ethyleneglycol) (abbreviated herein to PEG) or a derivativethereof. In certain embodiments, the antibody, antibody derivative orantibody fragment of this invention is an antibody fragment that is aFab′ or F(ab′)₂ fragment or a derivative thereof which is attached toPEG either on the heavy chain or the light chain or both. This Fab′ orF(ab′)₂ fragment thereof may be human or humanized.

Accordingly, in certain embodiments of this invention, the anti-CD154antibodies of this invention are antibodies that are modified bycovalent attachment of functional moieties such as water-solublepolymers, such as poly(ethyleneglycol), copolymers ofpoly(ethyleneglycol) and poly(propyleneglycol), carboxymethyl cellulose,dextran, poly(vinyl alcohol), poly(vinylpyrrolidone) orpoly(proline)—all of which are known to exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified proteins. See, e.g., Abuchowski et al. 1981 in“Enzymes as Drugs”, Holcenberg et al. (ed.), Wiley-Interscience, NewYork, N.Y., 367-383 (1981); Anderson, W. F. 1992, “Human Gene Therapy”,Science 256:808-813; Newmark et al. 1982, J. Appl. Biochem. 4:185-189;Katre et al. 1987, Proc. Natl. Acad. Sci. USA 84:1487-1491.

In some embodiments, antibodies of the present invention are antibodiesattached to functional moieties such as poly(ethyleneglycol) (PEG)moieties. In one particular embodiment, the antibody is an antibodyfragment and the PEG molecules may be attached through any availableamino acid side-chain or terminal amino acid functional group located inthe antibody fragment, for example, any free amino, imino, thiol,hydroxyl or carboxyl group. Such amino acids may occur naturally in theantibody fragment or may be engineered into the fragment usingrecombinant DNA methods (see for example U.S. Pat. Nos. 5,219,996;5,667,425; WO 98/25971). In another embodiment, a Fab fragment of thisinvention is modified by the addition to the C-terminal end of its heavychain one or more amino acids to allow the attachment of a functionalmoiety. Preferably, the additional amino acids form a modified hingeregion containing one or more cysteine residues to which the functionalmoiety may be attached. Multiple sites can be used to attach two or morePEG molecules.

In certain aspects of this invention, PEG molecules are covalentlylinked through a thiol group of at least one cysteine residue located inan antibody fragment of this invention. Each PEG molecule attached tothe modified antibody fragment may be covalently linked to the sulphuratom of a cysteine residue located in the fragment. The covalent linkagewill generally be a disulphide bond or, in particular, a sulphur-carbonbond. Where a thiol group is used as the point of attachmentappropriately activated functional moieties, for example thiol selectivederivatives such as maleimides and cysteine derivatives, may be used. Anactivated PEG may be used as the starting material in the preparation ofPEG-modified antibody fragments as described above. The activated PEGmay be any PEG containing a thiol reactive group such as anα-halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g.maleimide, a vinyl sulphone or a disulphide. In certain embodiments, ananti-CD154 antibody conjugate may comprise two PEG molecules with twomaleimide molecules. Starting materials may be obtained commercially(for example from Nektar, formerly Shearwater Polymers Inc., Huntsville,Ala., USA) or may be prepared from commercially available startingmaterials using conventional chemical procedures. Particular PEGmolecules include 20K methoxy-PEG-amine (obtainable from Nektar,formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA(obtainable from Nektar, formerly Shearwater).

In one preferred embodiment, an antibody of the invention is a modifiedFab fragment which is PEGylated, i.e. has PEG (poly(ethyleneglycol))covalently attached thereto, e.g. according to the method disclosed inEP 0948544 (see also “Poly(ethyleneglycol) Chemistry, Biotechnical andBiomedical Applications”, 1992, J. Milton Harris (ed), Plenum Press, NewYork, “Poly(ethyleneglycol) Chemistry and Biological Applications”,1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society,Washington D.C. and “Bioconjugation Protein Coupling Techniques for theBiomedical Sciences”, 1998, M. Aslam and A. Dent, Grove Publishers, NewYork, and Chapman, A., Advanced Drug Delivery Reviews 2002, 54:531-545).In one example PEG is attached to a cysteine in the hinge region. Inanother example, a PEG modified Fab fragment has a maleimide groupcovalently linked to a single thiol group in a modified hinge region. Alysine residue may be covalently linked to the maleimide group and toeach of the amine groups on the lysine residue may be attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab fragment may therefore be approximately 40,000 Da.

In another embodiment, the functional moiety is PEG and is attachedusing the methods described in WO 98/25971 and WO 04/72116, whereby alysyl-maleimide group is attached to the cysteine residue at theC-terminal end of the heavy chain, and each amino group of the lysylresidue has covalently linked to it a methoxypoly(ethyleneglycol)residue having a molecular weight of about 20,000 Da. The totalmolecular weight of the PEG attached to the antibody is thereforeapproximately 40,000 Da.

In another embodiment, the functional moiety is PEG and is attached to aF(ab)₂ fragment using the methods described in WO 98/25971 and WO04/072116, whereby a lysyl-dimaleimide group is attached to the cysteineresidue at the C-terminal end of each Fab heavy chain, and each aminogroup of the lysyl residue has covalently linked to it amethoxypoly(ethyleneglycol) residue having a molecular weight of about20,000 Da. The total molecular weight of the PEG attached to the F(ab)₂antibody is therefore approximately 40,000 Da.

In certain embodiments of this invention, the antibody of this inventionis a Fab′ antibody fragment, which may be fully human or humanized, andis PEGylated either in the heavy chain, the light chain or both. Inother embodiments, the antibody fragment, which may be fully human orhumanized, is PEGylated on one or both heavy chains, or on one or bothlight chains, or on both heavy and light chains.

Accordingly, in certain embodiments, an anti-CD154 antibody is aPEG-linked antibody (e.g., a PEG-linked human antibody) wherein the PEGis linked to the antibody at a cysteine or at a lysine residue. Incertain embodiments, the PEGylated anti-CD154 antibody has ahydrodynamic size of at least 24 kD. In other embodiments, the PEG mayvary in size from anywhere between 20 to 60 kD (inclusive). In furtherembodiments, the PEG-linked anti-CD154 antibody has a hydrodynamic sizeof at least 200 kD. In embodiments of the present invention where theanti-CD154 antibody is linked to a PEG moiety, the PEGylated anti-CD154antibody may have an increased in vivo half-life relative to ananti-CD154 antibody that lacks the PEG moiety.

In certain embodiments, this invention provides a CD154 binding proteincomprising a light chain sequence of SEQ ID NO: 15 and a heavy chainsequence of SEQ ID NO: 13, wherein the protein is PEGylated.

Other functional moieties that may be useful in improving the integrityand longevity of the antibodies of the present invention in vivo includepolypeptides. For example, the anti-CD154 antibodies or antibodyfragments of this invention may be modified to include a human serumalbumin (HSA) polypeptide. Such an antibody conjugate may exhibitincreased stabilization and increased serum half-life compared to anon-conjugated antibody or antigen-binding fragment. For example, incertain embodiments, an anti-CD154 antibody conjugated to HSA mayexhibit increased in vivo half-life relative to a non-conjugatedanti-CD154 antibody. The half-life (tα- or tβ-half life) of theHSA-conjugated antibody may be increased by 10%, 20%, 30%, 40%, 50% ormore. The tα-half life may be within the range of 0.25 minutes to 12hours, for example, while the tβ-half life may be within 12-48 hours,for example. The tα- or tβ-half life may preferably be at least 3 days,at least 7 days, at least 14 days, at least 21 days, at least 28 days,at least 1 month or more.

In some embodiments of this invention, the anti-CD154 antibodies of thisinvention are antibodies modified with a functional moiety by labelingwith a detectable marker, for example, a radioactive isotope, enzyme,dye or biotin, or other affinity reagent.

In some embodiments of this invention, the anti-CD154 antibodies of thisinvention are antibodies modified with a functional moiety by beingconjugated to a therapeutic agent, for example, a radioisotope orradionuclide (e.g., 111In or 90Y), toxin moiety (e.g., tetanus toxoid orricin), toxoid or chemotherapeutic agent (U.S. Pat. No. 6,307,026).

In some embodiments of this invention, the anti-CD154 antibodies of thisinvention are antibodies modified by being conjugated to an imagingagent. Imaging agents may include for example a labeling moiety (e.g.,biotin, fluorescent moieties, radioactive moieties, a histidine or myctag or other peptide tags) for easy isolation or detection.

Further examples of functional moieties for modification of orconjugation to anti-CD154 antibodies of the invention may includeserotoxins or cytotoxic agents including any agent that is detrimentalto (e.g. kills) cells. Examples include combrestatins, dolastatins,epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin,halichondrins, roridins, hemiasterlins, taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

Functional moieties useful in conjugation include, but are not limitedto, anti-folates (e.g. aminopterin and methotrexate), antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g. daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins, CC-1065, enedieyenes, neocarzinostatin), and anti-mitoticagents (e.g. vincristine and vinblastine). See Garnett, 2001, AdvancedDrug Delivery Reviews, 53:171-216 for further details.

Other functional moieties may include chelated radionuclides such as¹³¹I, ¹¹¹In, ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Ifidium¹⁹²,Tungsten¹⁸⁸/Rhenium¹⁸⁸, and ²¹¹astatine, or drugs such as but notlimited to alkylphosphocholines, topoisomerase I inhibitors, taxoids andsuramin.

Further functional moieties include proteins, peptides and enzymes.Enzymes of interest include, but are not limited to, proteolyticenzymes, hydrolases, lyases, isomerases, and transferases. Proteins,polypeptides and peptides of interest include, but are not limited to,immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, ordiphtheria toxin, a maytansinoid (for example, but not limited to, DM1),a protein such as insulin, tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor ortissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g. angiostatin or endostatin, angiogenin, gelonin, dolstatins,minor groove binders, bis-ido-phenol mustard, or a biological responsemodifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nervegrowth factor (NGF) or other growth factor.

Other functional moieties may include detectable substances useful, forexample, in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions that can be conjugated to antibodies for use asdiagnostics. Suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; suitableprosthetic groups include streptavidin, avidin and biotin; suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; suitable luminescent materials includeluminol; suitable bioluminescent materials include luciferase,luciferin, and aequorin; and suitable radioactive nuclides include ¹²⁵I,¹³¹I, ¹¹¹In and ⁹⁹Tc.

Nucleic Acids

In certain aspects, the present invention relates to nucleic acidsencoding CD154 binding proteins, e.g., anti-CD154 antibodies, of thepresent invention.

Accordingly, in certain embodiments the present invention relates to anisolated, recombinant and/or synthetic DNA molecule that comprises oneor more sequence(s) selected from the group consisting of SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO: 55, SEQ ID NO: 57,SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 67, SEQ ID NO: 70 and SEQ IDNO: 73.

In another aspect, the disclosure features an isolated nucleic acid thatcomprises one or more sequence(s) that encode a polypeptide thatincludes a sequence at least 80, 85, 90, 92, 94, 95, 96, 97, 98, 99, or100% identical to the sequence of a variable domain sequence of SEQ IDNO: 1, SEQ ID NO: 2; SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 14, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 54, SEQ ID NO: 56, SEQID NO: 58 or SEQ ID NO: 60 or a sequence that hybridizes (e.g., understringent conditions) to a nucleic acid encoding the sequence of avariable domain of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 29, SEQ ID NO: SEQ ID NO: 56 or SEQ ID NO: 60.

The nucleic acids of the invention may further include regulatorysequences (e.g., a promoter sequence, an untranslated 5′ region, and anuntranslated 3′ region) and/or vector sequences. For example, thenucleic acid constitutes a vector. In still further embodiments, theinvention relates to a host cell comprising the vector. The host cellmay produce the antibody so that the antibody exhibits reduced or noglycosylation (e.g., if an Fc region is present).

The present invention also relates to sequence variants of the nucleicacids described above. For example, the present invention includesnucleic acid sequences that are about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, 99.5%, 99.9% or 100% identical to anyof the sequences provided herein, including fragments thereof andcomplements thereto. The present invention also includes nucleic acidsthat vary from the sequences specifically provided herein due to thedegeneracy of the genetic code.

Further, the present invention includes sequences that specificallyhybridize to any of the nucleic acids provided herein. The term“specifically hybridizes” refers to the ability of a nucleic acidsequence to hybridize to at least 12, 15, 20, 25, 30, 35, 40, 45, 50 or100 consecutive nucleotides of a sequence provided herein, or a sequencecomplementary thereto, such that it has less than 15%, preferably lessthan 10%, and more preferably less than 5% background hybridization to acontrol nucleic acid (e.g., a non-specific DNA or DNA other than thespecific antibody sequence provided herein). A variety of hybridizationconditions may be used to detect specific hybridization, and thestringency is determined primarily by the wash stage of thehybridization assay. Generally high temperatures and low saltconcentrations give high stringency, while low temperatures and highsalt concentrations give low stringency. Low stringency hybridization isachieved by washing in, for example, about 2.0×SSC at 50° C., and highstringency is achieved with about 0.2×SSC at 50° C.

The nucleic acids encoding the CD154 binding proteins of the presentinvention may comprise leader or signal sequences. Leader and signalsequence can vary and may be substituted with an alternative leadersequence, and it is understood that, in certain embodiments, CD154binding proteins of the present invention comprise sequences without aleader sequence. Any suitable alternative leader or signal sequences maybe used.

Host Cells

The present invention relates to host cells engineered to express any ofthe DNA molecules provided in FIGS. 2-8, 10, 11 and 13-16, includingsequence variants thereof.

Host cells expressing the CD154 binding proteins, e.g., anti-CD154antibodies, of this invention are also provided. Whether a bindingprotein or an antibody, it may comprise only one chain, in which caseonly the DNA sequence encoding that polypeptide chain needs to be usedto transfect the cells. For the production of antibodies comprising twochains, the cell line may be transfected with two vectors.Alternatively, when appropriate, a single vector may encode both chainsequences, e.g. the light and heavy chain of an anti-CD154 antibody, andvariations depending on the particular antibody structure to beexpressed. The host cells may be, for example, prokaryotic cells such asE. coli, or other microbial cells, or eukaryotic cells including but notlimited to mammalian cells such as human, mouse, monkey, rabbit, goat,hamster, or rat cells, insect cells, avian cells, plant cells and lowereukaryotic cells such as fungal cells (see below). It is understood thatthe host cell machinery is responsible for glycosylating recombinantlyexpressed proteins and thus particular glycosylation patterns can beselected to further alter effector function of antibodies of theinvention.

In some embodiments of this invention, the host cells useful forpracticing the invention may be, for example, (1) bacterial cells, suchas E. coli, Caulobacter crescentus, Streptococci, Staphylococci,Streptomyces species and Bacillus subtilis cells, and Salmonellatyphimurium; (2) fungal cells and Aspergillus cells, yeast cells, suchas Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,Pichia methanolica, other Pichia species, and K. lactis; (3) insect celllines, such as those from Spodoptera frupperda—e.g., Sf9 and Sf21 celllines, and EXPRESSF® (a cell line from Spodoptera frugiperda) cells(Protein Sciences Corp., Meriden, Conn., USA)—Drosophila S2 cells, andTrichoplusia ni HIGH FIVE® (a cell line from the parental Trichoplusiani cell line) Cells (Invitrogen, Carlsbad, Calif., USA); (4) mammaliancells; or (5) plant cells.

Accordingly, the CD154 binding proteins, e.g., anti-CD154 antibodies, ofthis invention may be produced in any available prokaryotic oreukaryotic host cells capable of being engineered to express exogenousnucleic acid sequences. Lower eukaryotic host cells that may be used toproduce anti-CD154 antibodies of the present invention include thosecells described in the art (see, e.g., WO 02/00879, WO 03/056914, WO04/074498, WO 04/074499, Choi et al., 2003, PNAS, 100: 5022-5027;Hamilton et al., 2003, Nature, 301: 1244-1246 and Bobrowicz et al.,2004, Glycobiology, 14: 757-766).

Typical mammalian cells include COS1 and COS7 cells, Chinese hamsterovary (CHO) cells, NS0 myeloma cells, NIH 3T3 cells, 293 cells, HEPG2cells, HeLa cells, C127, 3T3, BHK, Bowes melanoma cells, L cells, MDCK,HEK293, WI38, murine ES cell lines (e.g., from strains 129/SV, C57/BL6,DBA-1, 129/SVJ), K562, Jurkat cells, and BW5147. The invention thusprovides cells that express the antibodies of the present invention,including but not limited to hybridoma cells, B cells, plasma cells, aswell as mammalian and human host cells recombinantly modified to expressthe antibodies of the present invention (e.g., adult embryonic stemcells). Other useful mammalian cell lines are well known and readilyavailable from the American Type Culture Collection (“ATCC”) (Manassas,Va., USA) and the National Institute of General Medical Sciences (NIGMS)Human Genetic Cell Repository at the Coriell Cell Repositories (Camden,N.J., USA). These cell types are only representative, and this list isnot meant to be an exhaustive list.

Among other considerations, some of which are described above, a hostcell may be chosen for its ability to process the expressed anti-CD154antibody in the desired fashion. In addition to modified glycosylationand aglycosylation, such post-translational modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,carboxymethylation, phosphorylation, lipidation, and acylation.

In another embodiment of this invention, the anti-CD154 antibodies ofthis invention are prepared by cell free translation or synthesized invitro. The genes that encode for these proteins may be synthesized invitro.

In another embodiment, the anti-CD154 antibodies of this invention areproduced in bioreactors containing the antibody-expressing cells, inorder to facilitate large scale production.

In another embodiment, the CD154 binding proteins or anti-CD154antibodies of this invention are produced in genetically engineered ortransgenic mammals (e.g., goats, cows, sheep) that express the antibodyin milk, in order to facilitate large scale production of the anti-CD154antibodies (U.S. Pat. No. 5,827,690; Pollock et al. 1999. J. Immunol.Meth. 231(1-2):147-57).

Therapeutic Methods

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, or a pharmaceutical composition comprising theantibody, is capable of inhibiting an immune response in a subject. Theantibody of this invention, or pharmaceutical composition of theinvention, is administered to the subject in an effective inhibitingamount.

In certain embodiments, an “effective inhibiting amount” of ananti-CD154 antibody, or pharmaceutical composition comprising theantibody, is any amount which is effective to inhibit the CD154-CD40interaction in the subject to whom it is administered. Methods ofdetermining an “inhibiting amount” are well known to those skilled inthe art and depend upon factors including, but not limited to: the typeof subject involved, the size and age of the subject and thepharmacokinetic properties of the particular therapeutic agentdelivered.

In another specific embodiment of this invention, a CD154 bindingprotein, e.g., an anti-CD154 antibody, of this invention, or apharmaceutical composition comprising the antibody, is capable ofinhibiting the immune response by inhibiting the CD154-CD40 interaction.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting inflammation. For thepurposes of this invention, inflammatory responses are characterized byredness, swelling, heat and pain, as consequences of capillary dilationwith edema and migration of phagocytic leukocytes. Some examples ofinflammatory responses include: arthritis, contact dermatitis, hyper-IgEsyndrome, inflammatory bowel disease, allergic asthma, and idiopathicinflammatory disease. Idiopathic inflammatory disease includes, forexample, psoriasis and lupus (e.g., systemic lupus erythematosus (SLE),drug-induced lupus erythematosus, and lupus nephritis). See, e.g.,Gallin 1989, Fundamental Immunology, Chapter 26, Raven Press, 2d Ed.,pp. 721-733, New York. This invention provides a method of treating orpreventing a symptom of systemic lupus erythematosus (SLE) in anindividual, the method comprising administering a monovalent CD154binding protein, e.g., a monovalent anti-CD154 antibody, to anindividual in an amount effective to treat or prevent a symptom of SLE.

Some examples of arthritis include: rheumatoid arthritis, non-rheumatoidinflammatory arthritis, arthritis associated with Lyme disease andinflammatory osteoarthritis. Some examples of idiopathic inflammatorydisease include: psoriasis and systemic lupus.

In one embodiment of this invention, an anti-CD154 antibody, or apharmaceutical composition comprising the antibody, is capable ofinhibiting rejection by the subject of a transplanted organ.

In a more specific embodiment of this invention, a CD154 bindingprotein, e.g., an anti-CD154 antibody of this invention, or apharmaceutical composition comprising the antibody, is capable ofinhibiting rejection by the subject of a transplanted heart, kidney,liver, skin, pancreatic islet cells or bone marrow.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, or a pharmaceutical composition comprising theantibody, is capable of inhibiting graft-vs-host disease in a subject.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, or a pharmaceutical composition comprising theantibody, is capable of inhibiting allergic responses in a subject—forexample, hay fever or an allergy to penicillin or other drugs.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting the autoimmuneresponse in a subject suffering from autoimmune disease. In someembodiments, the autoimmune response is associated with or is derivedfrom a condition selected from the group consisting of: rheumatoidarthritis, myasthenia gravis, systemic lupus erythematosus, Graves'disease, idiopathic thrombocytopenia purpura, hemolytic anemia, diabetesmellitus, inflammatory bowel disease, Crohn's disease, multiplesclerosis, psoriasis, drug-induced autoimmune diseases, or drug-inducedlupus. In certain embodiments, the autoimmune response is associatedwith or derived from systemic lupus erythematosus.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting an autoimmune responsein a subject suffering from an autoimmune response which is derived froman infectious disease.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting an autoimmune responsein a subject suffering from an autoimmune response which is derived fromReiter's syndrome, spondyloarthritis, Lyme disease, HIV infection,syphilis, or tuberculosis.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting fibrosis in a subject.

Some examples of fibrosis include pulmonary fibrosis or fibroticdisease. Some examples of pulmonary fibrosis include pulmonary fibrosissecondary to adult respiratory distress syndrome, drug-induced pulmonaryfibrosis, idiopathic pulmonary fibrosis, or hypersensitivitypneumonitis. Some examples of fibrotic diseases include: Hepatitis-C;Hepatitis-B; cirrhosis; cirrhosis of the liver secondary to a toxicinsult; cirrhosis of the liver secondary to drugs; cirrhosis of theliver secondary to a viral infection; and cirrhosis of the liversecondary to an autoimmune disease.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting gastrointestinaldisease. Some examples of gastrointestinal disease include: esophagealdysmotility, inflammatory bowel disease (including Crohn's disease andulcerative colitis), gastritis, collagenous colitis (includinglymphocytic colitis and microscopic colitis), celiac disease (alsocalled gluten enteropathy, celiac sprue, or gluten intolerance), andscleroderma.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting vascular disease. Someexamples of vascular disease include: atherosclerosis, renal arterydisease, lymphedema, ischemic disorders, and reperfusion injury. Alsoincluded are collagen vascular/immune complex diseases such as systemiclupus erythematosus or cryoglobulinemia.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting the proliferation of Tcell tumor cells in a subject suffering from a T cell cancer—e.g., a Tcell leukemia or lymphoma. Such an anti-CD154 antibody, or apharmaceutical composition comprising the antibody, may be administeredto the subject in an amount effective to inhibit the proliferation of Tcell tumor cells in that subject.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of inhibiting viral infection of theT cells of a subject by the human T-cell lymphotropic virus type 1 (HTLVI). Such an anti-CD154 antibody or a pharmaceutical compositioncomprising the antibody may be administered to the subject in an amounteffective to inhibit viral infection.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody, of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of imaging tumor cells or neoplasticcells in a subject that express a CD154 protein to which the antibody ofthis invention specifically binds. A method for imaging tumor cells orneoplastic cells in a subject comprises the steps of: administering tothe subject an effective amount of an anti-CD154 antibody of thisinvention, or a composition comprising it, under conditions permittingthe formation of a complex between the antibody and a protein on thesurface of tumor cells or neoplastic cells; and imaging anyantibody/protein complex formed, thereby imaging any tumor cells orneoplastic cells in the subject.

In one embodiment of this invention, a CD154 binding protein, e.g., ananti-CD154 antibody of this invention, or a pharmaceutical compositioncomprising the antibody, is capable of detecting the presence of tumorcells or neoplastic cells in a subject that express a CD154 protein towhich the antibody of this invention specifically binds. One such methodfor detecting the presence of tumor cells or neoplastic cells in asubject comprises the steps of: administering to the subject aneffective amount of an anti-CD154 antibody of this invention, or apharmaceutical composition comprising it, under conditions permittingthe formation of a complex between the antibody and a protein; clearingany unbound imaging agent from the subject; and detecting the presenceof any antibody/protein complex formed, the presence of such complexindicating the presence of tumor cells or neoplastic cells in thesubject.

Pharmaceutical Compositions

This invention provides pharmaceutical compositions comprising a CD154binding protein, e.g., an anti-CD154 antibody, as described in thisinvention.

In one embodiment of this invention, the pharmaceutical compositioncomprises at least one CD154 binding protein, e.g., anti-CD154 antibody,of this invention.

In one embodiment of this invention, an aglycosyl anti-CD154 antibody(or other anti-CD154 antibody with reduced effector function) of theinvention, or pharmaceutical composition comprising the antibody, iscapable of binding to a CD154 antigen (e.g., the CD154 antigen that isspecifically bound by aglycosyl hu5c8 produced by the cell line havingATCC Accession No. PTA-4931), and wherein the aglycosyl anti-CD154antibody is characterized by having a mutation of N298Q (N297 using EUKabat numbering) and which further exhibits reduced effector function asdescribed elsewhere herein.

In certain embodiments of this invention, an aglycosyl anti-CD154antibody (or other anti-CD154 antibody with reduced effector function),or pharmaceutical composition comprising the antibody, does not bind toan effector receptor. In a more specific embodiment, an aglycosylanti-CD154 antibody, or pharmaceutical composition comprising theantibody, is capable of binding to the CD154 protein that isspecifically bound by aglycosyl hu5c8 produced by the cell line havingATCC Accession No. PTA-4931, and wherein the aglycosyl anti-CD154antibody or pharmaceutical composition does not bind to an effectorreceptor.

In a specific embodiment of this invention, an aglycosyl anti-CD154antibody (or other anti-CD154 antibody or CD154 binding protein withreduced effector function), or pharmaceutical composition comprising theantibody, does not cause thrombosis, including thromboembolic events. Ina more specific embodiment of this invention, an aglycosyl anti-CD154antibody or pharmaceutical composition comprising the antibody iscapable of binding to the CD154 protein that is specifically bound byaglycosyl hu5c8 produced by the cell line having ATCC Accession No.PTA-4931, and wherein the aglycosyl anti-CD154 antibody orpharmaceutical composition does not cause thrombosis.

In another embodiment of this invention, the pharmaceutical compositionsmay further comprise any one or more of a pharmaceutically acceptablecarrier, an adjuvant, a delivery vehicle, a buffer and/or a stabilizer.Exemplary techniques for formulation and administration of theantibodies of the present invention may be found, for example, in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition.

In a more particular embodiment of this invention, the pharmaceuticallyacceptable carrier is phosphate buffered saline, physiological saline,water, or a citrate/sucrose/Tween formulation or emulsion—e.g.,oil/water emulsion.

In one embodiment of this invention, the pharmaceutical composition maybe delivered in a microencapsulation device so as to reduce or prevent ahost immune response against the composition. The binding agents, suchas antibodies or antibody fragments of this invention, may also bedelivered microencapsulated in a membrane, such as a liposome or otherencapsulated or immunoprotected delivery vehicle.

In one embodiment of this invention, the pharmaceutical composition maybe in the form of a sterile injectable preparation, for example, asterile injectable aqueous or oleaginous suspension. This suspension maybe formulated according to techniques known in the art using suitabledispersing, wetting, and suspending agents.

In one embodiment of this invention, the pharmaceutical composition maybe delivered orally, topically or intravenously. When administeredsystemically, the therapeutic composition should be sterile,substantially pyrogen-free and in a parenterally acceptable solutionhaving due regard for pH, isotonicity, and stability. For example, apharmaceutical preparation is substantially free of pyrogenic materialsso as to be suitable for administration as a human therapeutic. Theseconditions are known to those skilled in the art.

In a more specific embodiment of this invention, for oraladministration, the pharmaceutical composition is formulated in asuitable capsule, tablet, aqueous suspension or solution. Solidformulations of the compositions for oral administration can containsuitable carriers or excipients, such as corn starch, gelatin, lactose,acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalciumphosphate, calcium carbonate, sodium chloride, or alginic acid.Disintegrators that can be used include, without limitation,microcrystalline cellulose, corn starch, sodium starch glycolate, andalginic acid. Tablet binders that can be used include acacia,methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone)(POVODONE®, hydroxypropyl methylcellulose, sucrose, starch, andethylcellulose. Lubricants that can be used include magnesium stearates,stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.

In a more specific embodiment of this invention, for topicalapplications, the pharmaceutical compositions may be formulated in asuitable ointment. Some examples of formulations of a composition fortopical use include: drops, tinctures, lotions, creams, solutions, andointments containing the active ingredient and various supports andvehicles.

In one embodiment of this invention, a topical semi-solid ointmentformulation typically comprises a concentration of the active ingredientfrom about 1 to 20%, e.g., 5 to 10%, in a carrier, such as apharmaceutical cream base.

In one embodiment of this invention, pharmaceutical compositions forinhalation and transdermal compositions can also readily be prepared.The therapeutic composition can be administered through the nose orlung, for example, as a liquid or powder aerosol (lyophilized).

In one embodiment of this invention, liquid formulations of apharmaceutical composition for oral administration prepared in water orother aqueous vehicles can contain various suspending agents such asmethylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan,acacia, polyvinylpyrrolidone, and polyvinyl alcohol. Liquid formulationsof pharmaceutical compositions of this invention can also includesolutions, emulsions, syrups and elixirs containing, together with theactive compound(s), wetting agents, sweeteners, and coloring andflavoring agents. Various liquid and powder formulations of thepharmaceutical compositions can be prepared by conventional methods forinhalation into the lungs of the mammal to be treated.

In one embodiment of this invention, liquid formulations of apharmaceutical composition for injection can comprise various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols—i.e.,glycerol, propylene glycol, liquid polyethylene glycol, and the like. Insome embodiments, the composition includes a citrate/sucrose/Tweencarrier. For intravenous injections, water soluble versions of thecompositions can be administered by the drip method, whereby apharmaceutical formulation containing the antifungal agent and aphysiologically acceptable excipient is infused. Physiologicallyacceptable excipients can include, for example, 5% dextrose, 0.9%saline, Ringer's solution or other suitable excipients. A suitableinsoluble form of the composition can be prepared and administered as asuspension in an aqueous base or a pharmaceutically acceptable oil base,such as an ester of a long chain fatty acid—e.g., ethyl oleate.

In one embodiment of this invention, the pharmaceutical compositioncomprises from about 0.1 to 90% by weight (such as 1 to 20% or 1 to 10%)of an anti-CD154 antibody of this invention, in a pharmaceuticallyacceptable carrier.

In one embodiment of this invention, the optimal percentage of theanti-CD154 antibody of this invention in each pharmaceutical compositionvaries according to the formulation itself and the therapeutic effectdesired in the specific pathologies and correlated therapeutic regimens.Pharmaceutical formulation is well-established in the art. Conventionalmethods, known to those of ordinary skill in the art of medicine, can beused to administer the pharmaceutical composition to the subject.

Accordingly, pharmaceutical compositions of the present invention relateto extended release formulations. Extended release, or controlledrelease or slow release, refers to drug formulations that release anactive drug, such as a polypeptide or antibody drug, over a period oftime following administration to a subject. Extended release ofpolypeptide drugs, which can occur over a range of desired times (e.g.,minutes, hours, days, weeks or longer, depending upon the drugformulation) differs from standard formulations in which substantiallythe entire dosage unit is available for immediate absorption orimmediate distribution via the bloodstream. Extended releaseformulations may, in certain embodiments, result in a level ofcirculating drug from a single administration that is sustained, forexample, for 8 hours or more, 12 hours or more, 24 hours or more, 36hours or more, 48 hours or more, 60 hours or more, 72 hours or more 84hours or more, 96 hours or more, or even, for example, for 1 week or 2weeks or more, for example, 1 month or more. Extended releasecompositions may comprise a monovalent anti-CD154 antibody of thepresent invention. In further embodiments, the monovalent antibody is ahumanized or a fully human antibody. In further embodiments, theanti-CD154 antibody is an antibody with reduced effector function asdescribed herein.

In some embodiments of this invention, the pharmaceutical compositionfurther comprises an immunosuppressive or immunomodulatory compound. Forexample, such an immunosuppressive or immunomodulatory compound may beone of the following: an agent that interrupts T cell costimulatorysignaling via CD28, an agent that interrupts calcineurin signaling, acorticosteroid, an anti-proliferative agent, and an antibody thatspecifically binds to a protein expressed on the surface of immunecells, including but not limited to CD45, CD2, IL2R, CD4, CD8, RANK FcR,B7, CTLA4, TNF, LTβ, and VLA-4.

In some embodiments of this invention, the immunosuppressive orimmunomodulatory compound is tacrolimus, sirolimus, mycophenolatemofetil or its active form mycophenolic acid, mizorubine,deoxyspergualin, brequinar sodium, leflunomide, rapamycin or azaspirane.

In other embodiments of this invention, antibodies of this invention orpharmaceutical compositions comprising them may be included in acontainer, package or dispenser alone or as part of a kit with labelsand instructions for administration.

Administration and Delivery Routes

The anti-CD154 antibodies of this invention and pharmaceuticalcompositions of this invention may be administered to a subject in anymanner which is medically acceptable. For the purposes of thisinvention, “administration” means any of the standard methods ofadministering an antibody, antibody fragment or pharmaceuticalcomposition known to those skilled in the art, and should not be limitedto the examples provided herein.

In some embodiments of this invention, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention may beadministered to a subject by injection intravenously, subcutaneously,intraperitoneally, intramuscularly, intramedullarily,intraventricularly, intraepidurally, intraarterially, intravascularly,intra-articularly, intra-synovially, intrasternally, intrathecally,intrahepatically, intraspinally, intratumorally, intracranially, byenteral, intrapulmonary, transmucosal, intrauterine, or sublingualroutes of administration, or locally, e.g., at sites of inflammation ortumor growth.

In some embodiments of this invention, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention may beadministered to a subject orally or nasally, or by inhalation,ophthalmic, rectal, or topical routes.

In a more specific embodiment, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention may beadministered to a subject orally in the form of capsules, tablets,aqueous suspensions or solutions.

In a more specific embodiment, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention may beadministered to a subject topically by application of a cream, ointmentor the like.

In other embodiments of this invention, the anti-CD154 antibodies ofthis invention and pharmaceutical compositions of this invention mayalso be administered by inhalation through the use of a nebulizer, a drypowder inhaler or a metered dose inhaler.

In further embodiments of this invention, the anti-CD154 antibodies ofthis invention and pharmaceutical compositions of this invention may beadministered to a subject by sustained release administration, by suchmeans as depot injections of erodible implants directly applied duringsurgery or by implantation of an infusion pump or a biocompatiblesustained release implant into the subject.

In a more specific embodiment, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention may beadministered to a subject by injectable depot routes of administration,such as by using 1-, 3-, or 6-month depot injectable or biodegradablematerials and methods.

In a more specific embodiment, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention may beadministered to a subject by applying to the skin of the subject atransdermal patch containing the antibody, antibody derivative orpharmaceutical composition, and leaving the patch in contact with thesubject's skin, generally for 1 to 5 hours per patch.

In other embodiments of this invention, the anti-CD154 antibodies ofthis invention and pharmaceutical compositions of this invention may beadministered to a subject at any dose per body weight and any dosagefrequency that is medically acceptable. Acceptable dosage includes arange of between about 0.01 and 200 mg/kg subject body weight.

In any of the methods of using the antibodies or pharmaceuticalcompositions of this invention, the antibodies or pharmaceuticalcompositions may be administered to a subject in single or multipledoses daily, every 2, 3, 4, 5 or 6 days, weekly, monthly or any fractionor multiple thereof, and further may be administered to a subjectrepeatedly at intervals ranging from each day to every other month, asdetermined by the skilled practitioner.

In any of the methods of using the antibodies or pharmaceuticalcompositions of this invention, the binding proteins, antibodies orpharmaceutical compositions comprising them may be administered to asubject in need thereof at intervals for as long a time as medicallyindicated, ranging from days or weeks to the life of the subject. Infurther embodiments, the anti-CD154 antibodies of this invention, andpharmaceutical compositions of this invention may be administered to asubject repeatedly at intervals ranging from each day to every othermonth.

In one embodiment of this invention, the anti-CD154 antibodies of thisinvention and pharmaceutical compositions of this invention can beadministered in multiple doses per day, if desired, to achieve the totaldesired daily dose. The effectiveness of the method of treatment can beassessed by monitoring the subject for known signs or symptoms of adisorder.

For all embodiments of this invention, the dosage and dose rate of theanti-CD154 antibodies of this invention and pharmaceutical compositionsof this invention effective to produce the desired effects will dependon a variety of factors, such as the nature of the disease to betreated, the size and age of the subject, the goal of the treatment, thespecific pharmaceutical composition used, the pharmacokinetics of theactive agent, and the judgment of the treating physician.

Accordingly, anti-CD154 antibodies of this invention and pharmaceuticalcompositions of this invention, will be administered in an amounteffective to achieve their intended purpose. A therapeutically effectiveamount may refer to an amount of antibody effective to prevent,alleviate or ameliorate symptoms of disease or prolong the survival ofthe subject being treated. A therapeutically effective amount may beachieved by altering the dose and dosing schedule of administration ofthe subject antibodies.

The anti-CD154 antibodies of this invention and pharmaceuticalcompositions of this invention may be administered as a single dosagefor certain indications, such as preventing immune response to anantigen to which a subject is exposed for a brief time, such as anexogenous antigen administered on a single day of treatment. Examples ofsuch a therapy would include coadministration of the antibody fragmentof the invention along with a therapeutic agent, for example anantigenic pharmaceutical, an allergen or a blood product, or a genetherapy vector. In indications where antigen is chronically present,such as in controlling immune reaction to transplanted tissue or tochronically administered antigenic pharmaceuticals, the antibodyfragments or pharmaceutical compositions of the invention areadministered at intervals for as long a time as medically indicated,ranging from days or weeks to the life of the subject.

In any of the methods described herein, the antibodies or pharmaceuticalcompositions may be administered to a subject with a second agent. Incertain embodiments, the agent is a therapeutic agent, such as animmunomodulatory or immunosuppressive agent. The immunomodulatory orimmunosuppressive agent may be any of the following:

(a) an agent that interrupts T cell costimulatory signaling via CD28;(b) an agent that interrupts calcineurin signaling;(c) a corticosteroid;(d) an anti-proliferative agent; and(e) an antibody that specifically binds to a protein expressed on thesurface of immune cells, including but not limited to CD45, CD2, IL2R,CD4, CD8, RANK FcR, B7, CTLA4, TNF, LTβ, and VLA-4. Theimmunosuppressive or immunomodulatory compound may be, for example,tacrolimus, sirolimus, mycophenolate mofetil, mizorubine,deoxyspergualin, brequinar sodium, leflunomide, rapamycin or azaspirane.The antibody and second agent may be administered simultaneously orsequentially.

In some instances, it may be advantageous to administer one or morenucleic acids of the invention to a subject in need thereof. Therapeuticand diagnostic methods of the invention comprising the step ofadministering at least one nucleic acid of the invention according towell known methods are included in the scope of the present invention.

In one embodiment of this invention, the subject(s) that can be treatedby the above-described methods is an animal. Preferably, the animal is amammal. Examples of mammals that may be treated include, but are notlimited to, humans, non-human primates, rodents (including rats, mice,hamsters and guinea pigs), cows, horses, sheep, goats, pigs, dogs andcats. Preferably, the mammal is a human.

This invention may be better understood based on the Examples thatfollow. However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described herein.

EXAMPLES

The following examples illustrate the methods and products of thepresent invention. Suitable modifications and adaptations of thedescribed conditions and parameters normally encountered in the art ofmolecular biology that are apparent to those skilled in the art arewithin the spirit and scope of the present invention.

Example 1: Generating Anti-Human-CD154 Antibodies by Slam

Selected Lymphocyte Antibody Method (SLAM) (Babcook et al., 1996, Proc.Natl. Acad. Sci, 93, 7843-7848; WO 92/02551; de Wildt et al., 1997, J.Immunol. Methods, 207:61-67; and Lagerkvist et al., 1995, BioTechniques18:862-869) was used to identify and isolate anti-human CD154antibodies. SLAM enables cells producing high affinity antibodiesgenerated during in vivo immune responses to be isolated from anyspecies. The isolated individual antibody producing cells are thenclonally expanded followed by screening for those clones which produceanti-CD154 antibodies followed by the subsequent identification of thesequence of their variable heavy (V_(H)) and light (V_(L)) chain genes.A particular screening method is detailed in WO 04/051268. Thus, B cellsthat are positive for antibodies to human CD154 were isolated.

Several rat anti-human CD154 antibodies were identified and isolatedusing SLAM technology (FIG. 12). One of these antibodies, CA081 00342(the “342 antibody”), was humanized, as described in the followingexample. Its DNA and deduced amino acid sequences are shown in FIG. 2.The gene encoding this antibody was cloned.

Example 2: Humanization of CA081 00342—Creation of 342.G2

SLAM antibody 342 was humanized by grafting the CDRs onto human germlineframeworks. Alignments of the rat antibody (donor) sequence with thehuman germline (acceptor) frameworks are shown in FIG. 9, together withthe designed humanized sequence. The light chain germline acceptorsequence chosen was the human VK1 2-1-(1) 012 V-region plus JK1 J-region(V BASE, MRC Centre for Protein Engineering, UK; SEQ ID NOS: 35 and 36)(FIG. 3). The heavy chain germline acceptor sequence chosen was thehuman VH3 1-1 3-66 V-region plus JH4 J-region (V BASE, MRC Centre forProtein Engineering, UK; SEQ ID NOS: 37 and 38) (FIG. 3). In addition, adifferent VH acceptor framework was chosen, the human VH4 1-1 4-59sequence (SEQ ID NOS: 39 and 40) (FIG. 3). The CDRs grafted from thedonor to the acceptor sequence are as defined by Kabat (Kabat et al.,Sequence of proteins of immunological interest (1987), Bethesda Md.,National Institutes of Health, US), with the exception of CDR-H1 wherethe combined Chothia/Kabat definition is used (see WO91/09967). Forgrafts where only the CDRs were transferred from the donor antibody ontothe acceptor frameworks, versions were constructed in which key donorframework residues were also included. These residues were identifiedusing a method based on that described in WO 91/09967. For example, thelight chain graft VK1gL4 contains donor residues at positions 38, 71 and85; the heavy chain graft VH3 gH1 contains donor framework residues atpositions 24, 48, 49, 73 and 78; the heavy chain graft VH4 gH1 containsdonor framework residues at positions 48, 71 and 78. The sequences ofall these grafts are shown in FIG. 9.

Genes encoding these V-region sequences were designed and constructedusing standard molecular biology techniques by contract gene synthesiscompanies (Entelechon GbmH; DNA 2.0; Blue Heron). Modifications tocreate grafted variants were made using standardoligonucleotide-directed mutagenesis using PCR. The signal peptidesequences from the original rat antibodies were included in the originalgene designs, to permit expression using mammalian cell expressionvectors.

The grafted light chain genes were sub-cloned into a light chainexpression vector, which contains DNA encoding the human C-Kappaconstant region (Km3 allotype). The grafted heavy chain genes weresub-cloned into a human gamma-4 expression vector, which contains DNAencoding the human gamma-4 constant region containing the hingestabilizing mutation S241P (Angal et al., Mol Immunol. 1993,30(1):105-8). Any suitable expression vector may be used. The originalrat antibody genes were also sub-cloned into these vectors, creatingplasmids expressing the chimeric rat V-region/human C-region antibody asa benchmark in assays.

Co-transfection of light chain gene and heavy chain gene plasmids intoCHO cells enabled expression of IgG and analysis of human CD154 bindingby BIACORE® (an assay to measure molecular interactions).

In order to analyze expression in E. coli and activity as a monovalentFab′, the genes for key constructs were sub-cloned into expressionvector pTTOD (Fab) (WO 03/48208, WO 03/031475). Sub-cloning was achievedin a 2-stage process: first the VK gene fragment was cloned in as anEcoRV-BsiWI fragment; then the VH gene fragment was cloned in as aPvuII-XhoI fragment. This process fuses DNA encoding the signal peptidefrom the OmpA protein to the genes encoding both light and heavy chains,conferring secretion of translated protein to the bacterial periplasm.The resultant expression plasmids were transformed into E. coli K-12strain W3110 and used in induction experiments in small-scale shakeflasks and for high cell density fermentation.

TABLE 1 Affinity of 342 Fab constructs by BIACORE ® (an assay to measuremolecular interactions) (Purified Fab from 1 L fermentation) Graft ka(1/Ms) kd(1/s) KD(M) KD(pM) gL4gH1 1.53E+07 6.97E−05 4.55E−12 4.55

Selection of the optimal graft was made taking into account bothactivity in assay and expression levels of Fab in E. coli fermentation.An example of affinity determination by BIACORE® (an assay to measuremolecular interactions) is shown in Table 1. On this basis graft gL4gH1was chosen.

A plasmid was made encoding a Fab′ version of the graft gL4gH1. The DNAsequence of the insertion insert of this graft is shown in FIG. 8 (SEQID NO: 41). FIG. 8 also provides the sequence of an insert (SEQ ID NO:28) for expression of a Fab fragment, which can be used to make a F(ab)₂fragment.

Example 3: Creation of Aglycosylated HU5C8 and HU342 Antibodies bySite-Directed Mutagenesis

Aglycosylated hu5c8 and hu342 antibodies used in subsequent experimentswere created using standard recombinant DNA techniques. Aglycosylatedhu5c8 was made substantially as described in US2006/0193856, except forsubstitution of the huIgG4 Fc domain for the IgG1 Fc domain previouslyused, in order to further reduce effector function. The kappa lightchain sequence of aglycosylated hu5c8 is shown in FIG. 13 (SEQ ID NO:62, SEQ ID NO: 63 and SEQ ID NO: 64). The heavy chain of aglycosylatedhu5c8 contains two mutations made by site-directed mutagenesis in theCH2 (T299A, Kabat EU) and hinge (S228P, Kabat EU) domains (FIG. 14; SEQID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67). The T299A mutation modifiesthe N-glycosylation site in the CH2 domain so that it is no longer asubstrate for N-glycosylation enzymes, rendering the moleculeaglycosylated.

The aglycosylated hu342 antibody was derived from the 342 Fab fragmentvector. This sequence was modified by addition of human signal sequencesand the appropriate human constant domain sequences. The aglycosylatedhu342 antibody also comprises a huIgG4 Fc domain. The kappa light chainsequence of aglycosylated hu342 is shown in FIG. 15 (SEQ ID NO: 68, SEQID NO: 69 and SEQ ID NO: 70). The heavy chain of aglycosylated hu342contains two mutations made by site-directed mutagenesis in the CH2(T299A, Kabat EU) and hinge (S228P, Kabat EU) domains (FIG. 16; SEQ IDNO: 71, SEQ ID NO: 72 and SEQ ID NO: 73). The T299A mutation modifiesthe N-glycosylation site in the CH2 domain so that it is no longer asubstrate for N-glycosylation enzymes, rendering the moleculeaglycosylated. Both aglycosylated hu5c8 and hu342 were stably expressedin CHO cells.

Example 4: Binding to CD154: Binding Affinity Measurements

The BIACORE® (an assay to measure molecular interactions) technologymonitors the binding between biomolecules in real time and without therequirement for labeling. One of the interactants, termed the ligand, iseither immobilized directly or captured on the immobilized surface whilethe other, termed the analyte, flows in solution over the capturedsurface. The sensor detects the change in mass on the sensor surface asthe analyte binds to the ligand to form a complex on the surface. Thiscorresponds to the association process. The dissociation process ismonitored when the analyte is replaced by buffer. In the affinityBIACORE® assay, the ligand is an anti-CD154 antibody such as 342antibody and the analyte is the extracellular domain of human CD154.Details of the method are as follows:

Instrument: BIACORE® 3000, BIACORE® AB, Uppsala, Sweden.

Sensor chip: CMS (research grade) Catalogue Number: BR-1001-14, BIACORE®AB, Uppsala, Sweden. Chips were stored at 4° C.BIAnormalising solution: 70% (w/w) Glycerol. Part of BIAmaintenance KitCatalogue Number: BR-1002-51, BIACORE® AB, Uppsala, Sweden. TheBIAmaintenance kit was stored at 4° C.

Amine Coupling Kit: Catalogue Number: BR-1000-50, BIACORE® AB, Uppsala,Sweden.

Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Madeup to 75 mg/mL in distilled water and stored in 200 μL aliquots at −70°C. N-Hydroxysuccinimide (NHS). Made up to 11.5 mg/mL in distilled waterand stored in 200 μL aliquots at −70° C. 1 M Ethanolaminehydrochloride-NaOH pH 8.5. Stored in 200 μL aliquots at −70° C.Buffers: Running buffer is HBS-EP (being 0.01 M HEPES pH 7.4, 0.15 MNaCl, 3 mM EDTA, 0.005% Surfactant P20). Catalogue Number: BR-1001-88,BIACORE® AB, Uppsala, Sweden. Buffer stored at 4° C. Immobilizationbuffer is Acetate 5.0 (being 10 mM sodium acetate pH 5.0). Cataloguenumber: BR-1003-51, BIACORE® AB, Uppsala, Sweden. Buffer stored at 4° C.Ligand capture: Affinipure F(ab′)₂ fragment goat anti-human IgG, Fab′fragment specific (Catalogue number: 109-006-097) or Fc fragmentspecific (Catalogue number: 109-006-098), Jackson ImmunoResearch Inc(Pennsylvania, USA). Reagents stored at 4° C.Ligand: anti-CD154 antibodies.Analyte: Recombinant extracellular domain of human CD154. Material wasprepared at 2 mg/mL (40 pM) in phosphate-buffered saline, stored at 4°C., and diluted in HBE-EP running buffer for the assays. Typically CD154was diluted from ˜1 nM to −100 pM by doubling dilutions for the affinityassay.Regeneration Solution: 40 mM HCl prepared by dilution with distilledwater from an 11.6 M stock solution (BDH, Poole, England. Cataloguenumber: 101254H). 5 mM NaOH prepared by dilution with distilled waterfrom a 50 mM stock solution. Catalogue number: BR-1003-58, BIACORE® AB,Uppsala, Sweden.Assay Method: BIA (Biamolecular Interaction Analysis) was performedusing a BIACORE® 3000 (BIACORE® AB). Affinipure F(ab′)₂ fragment goatanti-human IgG, Fc- or Fab′-fragment specific (Jackson ImmunoResearch)were immobilized on a CMS Sensor Chip via amine coupling chemistry to acapture level of ≈6000 response units (RUs). HBS-EP buffer (10 mM HEPESpH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, BIACORE® AB) wasused as the running buffer with a flow rate of 10 μl/min. Anti-CD154antibodies or Fab fragments were used at concentrations such that, oncecaptured by the immobilized anti-human IgG-Fc (or anti-human IgG Fab′)surface, gave a signal of ≈200 RUs. Human CD154 was titrated over thecaptured antibody, at various concentrations. 90 μL of CD154 wasinjected over the surface (association phase), followed by a 240 seconddissociation phase, all at a flow rate of 30 μL/min. The surface wasregenerated by two 10 μL injections of 40 mM HCl, followed by a 5 μLinjection of 5 mM NaOH at a flowrate of 10 μL/min. Backgroundsubtraction binding curves were analyzed using the BIAevaluationsoftware (version 3.2) following standard procedures. Kinetic parameterswere determined from the fitting algorithm.

This method may be used to assess the affinity for a CD154 protein, or afragment thereof, of any antibody, antibody derivative or antibodyfragment of this invention. Kd values obtained by BIACORE® foranti-CD154 antibodies isolated by SLAM are shown in FIGS. 12 and 18.

Example 5: Inhibition of CD40 Binding

A flow cytometry-based assay was used to assess the binding of labeledCD40 to CD154-expressing D1.1 cells. D1.1 Jurkat cells (American TypeCulture Collection) were maintained in RPMI 1640 medium (Gibco,31870-025) containing 10% (v/v) fetal calf serum (FCS), 2 mM glutamine(Invitrogen, 23030024), 1 mM sodium pyruvate (Invitrogen, 11360-039), 1%(v/v) D-(+)-glucose (Sigma, G8769) and 10 mM HEPES (Sigma, H0887). Attime of assay, 100,000 D1.1 cells were incubated in 100 μL RPMI 1640medium+10% FCS, in the presence or absence of serially dilutedanti-CD154 antibody for 15 minutes at room temperature. 5 μL of a 1:75dilution of hCD40-mFc-PE (Alexis Corp, ANC-504-050) was then added andincubated for a further 30 minutes at room temperature. After washingtwice in phosphate-buffered saline (PBS) containing 1% (w/v) bovineserum albumin (BSA fraction V, Serologicals Proteins Inc, 81-068-5) and0.02% (w/v) sodium azide (BDH, 103692K), cells were resuspended in 200μL PBS/1% BSA/0.02% sodium azide and flow cytometry was performed on aBecton Dickinson FACScan. The values for geometric mean fluorescence(FL2) were assessed in all cases. The inhibition of hCD40-mFc-PE bindingwas calculated relative to the signal in the absence of antibody (0%inhibition) and in the signal in the absence of hCD40-mFc-PE (100%inhibition), using the formula:

$\left\lbrack \frac{{0\% \mspace{14mu} {Inhibition}} - {\% \mspace{14mu} {Test}}}{{0\% \mspace{14mu} {Inhibition}} - {100\% \mspace{14mu} {Inhibition}}} \right\rbrack \times 100$

IC50 values from the data were obtained using XLfit as part of theActivity Base package.

The CD40 binding IC50 values for anti-CD154 antibodies isolated by SLAMare shown in FIGS. 12 and 18.

Example 6: Competition Binding Assay

A flow cytometry-based assay was used to assess the binding ofanti-CD154 antibody to CD154-expressing D1.1 cells. D1.1 Jurkat cells(American Type Culture Collection) were maintained in RPMI 1640 medium(Gibco, 31870-025) containing 10% (v/v) fetal calf serum (FCS), 2 mMglutamine (BioWhittaker, 17-605E) and 1 Penicillin-Streptomycin(Mediatech, 30002107). At time of assay, 100,000 D1.1 cells wereincubated in 10 mL PBS, 0.1% BSA, 0.02% sodium azide (FACS buffer), inthe presence or absence of serially diluted anti-CD154 antibody andbiotinylated anti-CD154 Fab (clone 342) for 2 hours at 4° C. The cellswere washed three times in FACS buffer with centrifugation at 290×g for3 minutes in between washes. A 1:500 dilution ofstreptavadin-R-phycoerythrin conjugate (Jackson Immunoresearch,016-110-084) in 150 μM FACS buffer was added and the cells wereincubated for one hour at 4° C. The cells were washed once and fixed inPBS with 3% formaldehyde at room temperature for 10 minutes. The cellswere resuspended in FACS buffer and run on a FACSCalibur (BD). Thevalues for geometric mean fluorescence (FL2) were assessed in all cases.Biotin 342 Fab binding was plotted against the concentration ofcompeting antibody to obtain sigmoidal inhibition curves that were fitto a four parameter curve fit using GraphPad Prism. The IC₅₀ valuesgenerated in this assay are shown in FIG. 18.

Example 7: ICAM-1 Upregulation Assay

The ability of the anti-CD154 antibodies to inhibit CD40L:CD40 cellsurface interactions was measured in an in vitro co-culture potencyassay. Ligation (e.g., binding) of CD40 with CD154 (CD40L) activates Blymphocytes resulting in an upregulation of CD54 (ICAM-1) on the cellsurface and this contact dependent CD40L:CD40 B cell activation can beblocked by anti-CD154. Briefly, D1.1 Jurkat T lymphoma cells (CRL-10915,American Type Culture Collection (ATCC), Manassas, Va., USA) expressingCD154 and Ramos 2G6.4C10 B lymphoma cells (CRL-1923, ATCC) expressingCD40 were co-cultured in a 37° C. incubator with 5% CO₂ overnight at a1:4 ratio with titrations of anti-CD154 Fabs or a control intact Ab(hu5C8). The assay was performed in 96-well round bottom plates at aconcentration of 1×10⁶ cells/ml in RPMI complete media (RPMI with 10%FBS, 1% L-glutamine, 1% sodium pyruvate and 10 mM HEPES pH 6.8, GibcoBRL, Rockville, Md., USA). The following day the cells were stained forone hour at 4° C. with CD20 FITC (#555622) and CD54 APC (#559771) fromBD Pharmingen (San Diego, Calif., USA) at a concentration of 1:100 and1:200 respectively in PBS containing 1% BSA and 0.1% sodium azide. Thecells were washed and fixed with 1% paraformaldehyde and analyzed on aFACScan Calibur Cytometer (BD Biosciences). The geometric meanfluorescence of the Ramos cells (double positive cells) versus theconcentration of anti-CD154 (CD40L) was fit to a 4-parameter curve usingDeltaGraph software (Red Rock Software, Salt Lake City, Utah, USA)(FIGS. 12 and 18). The IC50 values were used to determine the relativepotency of the anti-CD154 antibodies.

Example 8: Activity in Cynomolgus Monkey Model of Immune Response

The model used to demonstrate activity in vivo is described in Gobburuet al. (1998) J Pharmacol Exp Therapeutics 286: 925. Cynomolgus monkeysreceived single i.v. doses of either saline, hu5c8 antibody or a doseresponse of 342 Fab′-PEG, 4 hours prior to challenge with a single i.m.dose of 0.5 mL tetanus toxoid (TT). Each treatment group contained 3males and 3 females. On day 30, a second dose of inhibitor was given,and the animals were re-challenged with TT (the secondary response).Blood samples were taken at selected time periods for up to 50 days foranalysis of both IgG and IgM anti-TT titers (FIGS. 19 and 21). The datashow that 342 Fab′-PEG inhibits the IgG and IgM immune response to TT ina dose-dependent manner.

The IgG anti-TT titers in cynomolgus monkeys were also measured aftertreatment with a single dose (20 mg/kg for hu5c8, aglycosyl 5c8 andaglycosyl 342 and 40 mg/kg for 342 Fab′-PEG and 342 DFM-PEG) of variousforms of anti-CD154 antibodies (FIG. 20). Inhibition of the TT immuneresponse was observed with all antibodies evaluated.

Example 9: Fluorescence Activated Cell Sorting Cross-Blocking Assay

The binding properties of the anti-CD154 Fab′ fragments of the inventionwere studied by cross-blocking antibody assays. Briefly,CD154-expressing D1.1 Jurkat cells were incubated with either medium or10 μg/ml unlabeled first anti-CD154 Fab′ for 60 minutes at roomtemperature. After no washing, an optimal dilution of an Alexa Fluor488-labeled second anti-CD154 Fab′ (300 ng/ml) was added for 60 minutes.Cells were then washed and analyzed by flow cytometry. If the first andsecond Fab′ bind to the same epitope, the first Fab′ will competitivelyblock the binding of the second Fab′. If the two antibodies bind todifferent epitopes, the first Fab′ will not block the binding of thesecond Fab′. If the labeled second Fab′ tested is 342, it can bedemonstrated that the 342 Fab′ is cross-blocked by 338 (FIG. 23B), 381(FIG. 23D) and 335 (FIG. 23G) Fab's but is not cross-blocked by 295 (notshown), 402 (FIG. 23C), 300 (FIG. 23E), 303 (FIG. 23H) or 294 (FIG. 23F)Fab's. When the labeled Fab′ is hu5c8, cross-blocking by 338 (FIG. 24A),402 (FIG. 24B), 381 (FIG. 24C), 303 (FIG. 24D), 335 (FIG. 24E), 300(FIG. 24F) and 294 (FIG. 24G) Fab's can be demonstrated. Testing withA33 (an isotype-matched control antibody) confirms there is nonon-specific cross-blocking (FIGS. 23A and 24H). Antibodies 342 andhu5c8 competed with each other for CD154 binding regardless of which wasthe unlabeled (first) and labeled (second) Fab′.

Example 10: Biacore® Analysis of Antibody Binding

BIACORE® analysis of 342 and hu5c8 antibody binding to soluble CD154protein (sCD154) (extracellular domain; ECD) indicated that 342displaced hu5c8 binding to sCD154 when added to the sCD154 second (FIGS.25 A and B). 5c8 was not able to displace 342 from sCD154 when added tothe sCD154 second (FIG. 25 C). Antibody 338 behaved similarly to 342with respect to non-reciprocal results in hu5c8 competition assay.

Hu5c8 Fab can bind to a 342 full length (FL)/sCD154 complex (FIG. 25D).This result suggests that hu5c8 Fab does not compete with the 342 FLbinding site when 342 FL is added first. Without being bound by theory,these results suggest that binding of one or two arms of the sCD154trimer by 342 does not prevent hu5c8 binding to the “free” arm(s).

There is a slight (˜20RU) increase in binding when hu5c8 Fab is followedby 342 Fab′ (FIG. 25E). This result raises the possibility that eitherhu5c8 Fab′ has blocked binding of 342 Fab′ or that 342 Fab′ has replacedhu5c8 Fab′ on the captured sCD154.

Neither 342 Fab′ nor hu5c8 Fab′ bind to a CD40:sCD154 complex or affectthe dissociation of the complex in a protein assay. The fact thatneither Fab′ can bind the CD40:sCD154 complex suggests either that thecomplex uses all three sCD154 arms or that the complex stericallyhinders access of either Fab′ to a “free” arm.

Example 11: Human Platelet Activation Assay 1

Platelet aggregation can be measured using published assays (e.g.,Florian et al. (2005) Thrombosis Hemostasis 93: 1137). In one assay,platelets were washed from normal donor platelet-rich plasma inHEPES-buffered saline in BSA coated tubes and adjusted to 250,000 permicroliter (μ1). Washed platelets were then pipetted into anaggregometer cuvette (Chrono-Log 490D) and the trace signal wascalibrated to zero percent aggregation, using HEPES (assay) buffer forblanking. In this instrument the cuvette is maintained at 37.0° C. and asiliconized magnetic bar stirs the platelets at 1000 rpm. Fully formedimmune complexes (i.e., antibody plus recombinant human soluble CD40L(rhsCD40L), in 1:1 stoichiometry where rhsCD40L was treated as trimeric)were added to platelets and aggregation was assessed as a trace derivedfrom the solution's optical density. Specifically, 15 μl of immunecomplex was added to 285 μl of washed platelet suspension such thatafter adding to the cuvette, the final concentration of sCD40L was 10μg/ml and that of IgG antibody was 27.8 μg/ml or 16.7 μg/ml forFab′-PEG. The data show that aggregation occurs in the presence of theintact IgG anti-CD40L antibody hu5c8, but not with 342 Fab′-PEG (FIG.26).

Assay 2

In another assay described in WO 07/59332, platelet aggregation ismeasured by contacting platelets with a platelet activating agent (e.g.,adenosine diphosphate (ADP), collagen, thrombin, thromboxane, neurophilelastase, p-selectin, or convulxin), contacting the activated plateletswith an anti-CD154 antibody, and then contacting the activated plateletswith a cross-linking agent (e.g., soluble CD154 (sCD154), anti-human IgGantibody, anti-hFc antibody, RF, Fc receptor-positive accessory cell,soluble protein A, or soluble human Fc receptor). Aggregation is thenquantified by sedimentation of platelets, where sedimentation ofplatelets is indicative of aggregation of the platelets. The aggregationassay was performed on platelet rich plasma (PRP). Approximately 50 mLof whole blood was collected in aliquots in 4.5 mL vacutainer tubescontaining 0.5 mL of 3.8% sodium citrate. PRP was prepared bycentrifuging the anticoagulated blood at 200 g for 10 minutes andharvesting the supernatant. To perform the assay, the Biodata 4-channelplatelet aggregation profiler (PAP-4; Biodata Corp., Hatboro, Pa.) wasblanked using a cuvette containing only platelet poor plasma (PPP). A350 μL aliquot of PRP, containing approximately 2 to 5×10⁸/mL platelets,was added to a cuvette containing a stir bar. Anti-CD40L antibody, humanIgG, normal human serum, CD40-Fc, or anti-hFc were added in a totalvolume of 100 μL. The loaded cuvette was placed in the machine and thereaction components mixed prior to the addition of ADP.

Aggregation was initiated with the addition of sub-optimal concentrationof ADP in 50 μL (final concentration varies for each individual sample).The aggregation profiler has four ports, which can run simultaneously.An aggregation tracing was generated for each sample for four minutesfollowing the addition of ADP. At the end of the tracing, the instrumentcalculates the percentage of aggregation by comparing the transmissionof light through the sample to the transmission of light through the PPPblank. A titration was performed at the beginning of each experiment,and subsequent runs were performed at a suboptimal ADP concentration.

The results from this assay are shown in FIG. 27. PRP was obtained fromone healthy individual. Aggregation was induced with 0.75 μM ADP, whichwas determined to be suboptimal for this donor. A positive controlanti-CD154 antibody and a negative control hIgG were evaluated at 200μg/mL and sCD154 at 30 μg/mL. Anti-CD154 antibody or hIgG was mixed withrecombinant sCD154 for no less than 20 minutes prior to addition to thePRP-containing cuvette. Bars represent the means and standard deviationsof two data points. The results show that while negative control humanIgG (hIgG) and sCD154 together had no effect on platelet aggregation,the positive control anti-CD154 antibody enhanced platelet aggregation.The results from this assay using a positive and negative controldemonstrate that this assay may be used to compare the relative effectof the antibodies of the present invention on platelet aggregation.

To determine if the platelets express CD154 on their surface, theplatelets may be incubated with a biotin-conjugated anti-CD154 antibodyand the presence of surface CD154 determined by quantifying the boundbiotinylated anti-CD40L antibody. Accordingly, surface expression ofCD154 was evaluated after 1, 10, 20, 40, and 60 minutes of incubationwith or without 10 μM ADP. Surface expression of CD40L was detectable onADP-activated platelets as early as one minute after activation andincreased over time. The binding of biotin-conjugated anti-CD154antibody is specific for CD154, as preincubation of biotin-conjugatedanti-CD154 antibody with sCD154 inhibited binding to activatedplatelets. The amount of surface CD154 detected on inactivated(“resting”) platelets also increased over time. This phenomenon islikely attributable to the basal level of platelet activation under theexperimental conditions.

Example 12: Methods for Determining Altered Effector Function ofAglycosylated and Other Variant Antibodies

The following example describes assays useful for determining andcharacterizing effector function(s) of the aglycosylated and othermodified variant antibodies of the invention.

The effector function of the aglycosylated and other modified variantantibodies of the invention may be characterized by the antibodies'ability to bind an antigen and also bind an Fc receptor or a complementmolecule such as C1q. In particular, the FcγR binding affinities may bemeasured with assays based on the ability of the antibody to form a“bridge” between the CD154 antigen and a cell bearing an Fc receptor.The C1q binding affinity may be measured based on the ability of theantibody to form a “bridge” between the CD154 antigen and C1q. Theinteraction of the antibodies of the present invention with an FcR orwith complement can also be measured by the bead-based ALPHASCREEN® (amultiplex assay kit) technology PERKIN ELMER® (a biotechnology company).

Fc Receptor Binding Assays

The FcγR bridging assay may be performed by coating 96-well MaxisorbELISA plates (Nalge-Nunc Rochester, N.Y., USA) with recombinant solublehuman CD154 ligand (e.g., at a concentration of 1 μg/ml overnight at 4°C. in PBS; Karpusas et al. 1995 Structure 3(10): 1031-1039 and 3(12):1426 and Karpusas et al. 2001 Structure 9(4): 321-329). Titrations ofglycosylated or aglycosylated forms of anti-CD154 antibody are bound toCD154 for 30 minutes at 37° C., the plates are then washed, and thebinding of fluorescently labeled U937 (CD64+) cells are measured. TheU937 cells may be grown in RPMI medium with 10% FBS, 10 mM HEPES,L-glutamine, and penicillin/streptomycin, split 1:2, and activated forone day prior to the assay with 1000 units/ml of IFNγ to increase Fcreceptor (FcγRI) expression.

In another variation of the assay, the ability of the antibodies of theinvention to bind to, or rather, fail to bind to, yet another Fcreceptor, such as FcγRIII (CD16), may be performed using the abovebridging assay against fluorescently labeled human T cells (Jurkatcells) transfected with a CD16 expression construct. The ligand may beproduced by a monolayer of CD154-expressing Chinese Hamster Ovary (CHO)cells grown in 96-well tissue culture plates (Corning Life Sciences,Acton, Mass., USA). For example, the CHO-CD 154+ cells are seeded into96-well plates at 1×10⁵ cells/ml and grown to confluency in oc-MEM with10% dialyzed FBS, 100 nM methotrexate, L-glutamine, andpenicillin/streptomycin (Gibco-BRL, Rockville, Md., USA). The CD 16+Jurkat cells are grown in RPMI with 10% FBS, 400 pg/ml Geneticin, 10 mMHEPES, sodium pyruvate, L-glutamine, and penicillin/streptomycin(Gibco-BRL) and split 1:2 one day prior to performing the assay.

In the assays for both receptors, the Fc receptor-bearing cells may belabeled with 2′, 7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, Oreg., USA)for 20 minutes at 37° C. After washing to remove excess label, 1×10⁵ ofthe labeled cells are incubated in the assay for 30 minutes at 37° C.Unbound FcγR positive cells are removed by washing several times andplates are read on a microplate reader (Cytofluor 2350 FluorescentMicroplate Reader, Millipore Corporation, Bedford, Mass., USA) at anexcitation wavelength of 485 nm and an emission wavelength of 530 nm.

In addition to the assays described above, binding of the antibodies ofthe present invention to Fc receptors can be measured in a competitionformat using an AlphaScreen™ (Amplified Luminescent ProximityHomogeneous Assay; Perkin Elmer) or directly using Surface PlasmonResonance (BIACORE®). Biacore assays can monitor binding of analytereceptor to antibody captured on a protein A/G chip using a BIACORE®3000 instrument. BIACORE® is a well-established method forcharacterizing protein-protein interactions (Myszka 1997 Curr OpinBiotechnol. 8:50-57.; Malmborg & Borrebaeck 1995 J Immunol Methods183:7-13), and has been used successfully to measure the binding of IgGantibodies to FcγRIII (Galon et al., 1997 Eur J Immunol. 27:1928-1932).For example, protein A/G is coupled covalently to a sensor chip (e.g., aCMS sensor chip using NHS chemistry). A running buffer (e.g., HBS-EP(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20,BIACORE®)) and a chip regeneration buffer (e.g., Glycine 1.5 (10 mMglycine-HCl, pH 1.5,))BIACORE®)) are used in the assay. Variantanti-CD154 antibodies with reduced effector function and wildtype ornative anti-CD154 antibodies are diluted to 100 nM in running buffer(e.g., HBS-EP buffer) and are bound to the protein A/G chip for 5 min.Receptors are bound in the association phase in concentration series,followed by a dissociation phase with buffer. A cycle with no antibodyprovides a baseline response. Sensorgrams may be globally fit to a 1:1Langmuir binding model to obtain equilibrium dissociation constants(K_(D)s) using BIAevaluation software v4.1)(BIACORE®), for example.

AlphaScreen assays use untagged antibody to compete the interactionbetween biotinylated IgG bound to streptavidin donor beads andFcγR-His-GST bound to anti-GST acceptor beads. A wildtype or nativeanti-CD154 antibody (such as an IgG1 antibody) is biotinylated usingstandard methods and dialyzed in PBS. Anti-GST acceptor beads andstreptavidin donor beads are available from commercial vendors and canbe used at 20 μg/ml final concentration. FcγRI-His-GST,FcγRIIIa-His-GST, FcγRIIa-His-GST, or any other tagged FcR, in 1×assaybuffer (e.g., 25 mM HEPES, 100 mM NaCl, 0.1% BSA, 0.01% Tween-20, pH7.4) is distributed into each well of a 96-well plate to 0.5 nM finalconcentration. Wildtype or native anti-CD154 antibody, variantanti-CD154 antibody, or buffer is prepared as ½ log dilutions in 1×assay buffer, and aliquoted directly into each well. After briefcentrifugation, biotinylated wildtype anti-CD154 antibody (e.g., an IgG1antibody) in 1× assay buffer is added to each well to 5 nM finalconcentration. 100 μg/ml anti-GST acceptor beads are added to each well,and the plate is incubated in the dark for 1 hour at room temperature.100 μg/ml streptavidin donor beads are added to each plate, and afterbrief centrifugation the plate is incubated at room temperature for 1.5hours. Reaction samples are transferred to white opaque plates, andfluorescence is read in a FUSION® ALPHA-FP HT®, a universal microplatereader from PERKIN ELMER® (a biotechnology company). Data may benormalized to the highest signal (no competition) and fit to a one-sitecompetition model using nonlinear regression with the software GraphPadPrism (GraphPad Software), for example.

The skilled artisan may perform the above or similar assays to measurebinding of antibody variants to any FcR, including, for example,FcγRIIa.

C1q Binding Assays

The C1q binding assay may be performed by coating 96-well Maxisorb ELISAplates (Nalge Nunc, Rochester, N.Y., USA) with 50 μl recombinant solublehuman CD154 ligand (Karpusas et al. Structure, 15; 3 (12): 1426 (1995)at 10 μg/ml overnight at 4° C. in PBS. The wells are aspirated andwashed three times with wash buffer (PBS, 0.05% Tween 20) and blockedfor at least 1 hour with 200 μl/well of block/diluent buffer (0.1 MNa₂HPO₄, pH 7.0, 1 M NaCl, 0.05% Tween 20, 0.1% gelatin). The antibodyto be tested is diluted in block/diluent buffer starting at 15 μg/mlwith 3-fold dilutions. 50 μl is added per well, and the plates areincubated for 2 hours at room temperature.

After aspirating and washing as above, 50 μl/well of 2 μg/ml of Sigmahuman C1q (C0660) diluted in block/diluent buffer is added and incubatedfor 1.5 hours at room temperature. After aspirating and washing asabove, 50 μl/well of sheep anti C1q (Serotec AHP033), diluted 3,560-foldin block/diluent buffer, is added. After incubation for 1 hour at roomtemperature, the wells are aspirated and washed as above. 50 μl/well ofdonkey anti-sheep IgG HRP conjugate (Jackson ImmunoResearch,713-035-147) diluted to 1:10,000 in block/diluent is then added, and thewells are incubated for 1 hour at room temperature.

After aspirating and washing as above, 100 μl TMB substrate (420 μM TMB,0.004% H₂O₂ in 0.1 M sodium acetate/citric acid buffer, pH 4.9) is addedand incubated for 2 min before the reaction is stopped with 100 μl 2 Nsulfuric acid. The absorbance is read at 450 nm with a Softmax PROinstrument, and Softmax software is used to determine the relativebinding affinity (C value) with a 4-parameter fit.

An alternative C1q binding assay uses ELISA to determine anti-CD154binding to C1q but does not use CD154 as a bridge. Briefly, assay platesmay be coated overnight at 4° C. with a variant antibody or a parentantibody (control) in coating buffer. The plates may then be washed andblocked. Following washing, an aliquot of human C1q may be added to eachwell and incubated for 2 hours at room temperature. Following a furtherwash, 100 μl of a sheep anti-complement C1q peroxidase conjugatedantibody may be added to each well and incubated for 1 hour at roomtemperature. The plate may again be washed with wash buffer and 100 μlof substrate buffer containing OPD (0-phenylenediamine dihydrochloride(Sigma)) may be added to each well. The oxidation reaction, observed bythe appearance of a yellow color, may be allowed to proceed for 30minutes and stopped by the addition of 100 μl of 4.5 NH₂SO₄. Theabsorbance may then read at (492-405) nm.

An exemplary antibody variant is one that displays a “significantreduction in C1q binding” in this assay. A significant reduction may be,in some embodiments, about 100 μg/ml of the antibody variant displayingabout 50-fold or more reduction in C1q binding compared to 100 μg/ml ofa control antibody having a nonmutated IgG1 Fc region. In the mostpreferred embodiment, the polypeptide (i.e., antibody) variant does notbind C1q, i.e., 100 μg/ml of the antibody variant displays about100-fold or more reduction in C1q binding compared to 100 μg/ml of thecontrol antibody.

Complement Activation and CDC

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, e.g. as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996). Briefly, various concentrationsof the polypeptide (i.e., antibody) variant and human complement may bediluted with buffer. Cells which express the antigen to which thepolypeptide variant binds may be diluted to a density of about 1×10⁶cells/ml. Mixtures of polypeptide variant, diluted human complement andcells expressing the antigen may be added to a flat bottom tissueculture 96-well plate and allowed to incubate for 2 hours at 37° C. and5% CO₂ to facilitate complement mediated cell lysis. 50 μl of Alamarblue (Accumed International) may then be added to each well andincubated overnight at 37° C. The absorbance is measured using a 96-wellfluorometer with excitation at 530 nm and emission at 590 nm. Theresults may be expressed in relative fluorescence units (RFU). Thesample concentrations may be computed from a standard curve and thepercent activity as compared to nonvariant polypeptide is reported forthe polypeptide variant of interest.

Example 13: Mapping of 342 Antibody Binding Site on CD154 Protein

Experiments were carried out to identify the amino acid residues inhuman CD154 that are important in the binding of 342 using human-mousechimeric CD154 proteins. 342 binds with high affinity to human CD154 butdoes not bind to mouse CD154. Therefore, by mutating CD154 mouseresidues to those of the corresponding human residues and measuring thechange in the affinity of the mutated protein for 342, important bindingresidues can be identified. Six groups of mutants were selected wherehuman residues at selected regions were introduced into soluble mouseCD154 (marked 1-6 in FIG. 28). Non-mutated human and mouse soluble CD154(sCD154) proteins were also evaluated. The different sCD154 proteinswere used in BIACORE® experiments using 342 (in an IgG format)immobilized on the chip and with the sCD154 supernatants in the solutionphase. The results demonstrated that 342 binding only occurs with theintroduction of human Group 5 residues into mouse CD154.

Example 14: Competition ELISA Cross-Blocking Assay

To demonstrate cross-blocking of 342 and 5c8 Fab's, we used acompetition ELISA. In this assay, anti-Myc antibody 9E10 was coated ontoan ELISA plate. Myc-tagged CD154 was captured by this antibody. Adilution series of unlabeled 342 or 5c8 Fab′ was incubated with either 1nM biotin 342 Fab′ or 0.3 nM biotin 5c8 on the plate for two hours atroom temperature in PBS, 0.05% Tween-20, 1% BSA. The plate was washedand the amount of biotinylated 5c8 Fab bound was determined usingstreptavidin HRP as a secondary. The signal was graphed and fit usingeither a one site binding hyperbola or a two site binding hyperbolacurve fit in Prism software (GraphPad).

A titration of biotin 342 Fab′ on CD154 to determine the appropriateconcentration for cross-blocking analysis was performed (FIG. 29A). A 1nM concentration was found to be on the linear part of the curve. Atitration of biotin 5c8 Fab′ on CD154 to determine the appropriateconcentration for cross-blocking analysis was also performed (FIG. 29B).A 0.3 nM concentration was found to be on the linear part of the curve.In an experiment to analyze cross-blocking of biotin 342 and biotin 5c8Fab's by unlabeled 342 Fab′, 342 Fab′ inhibited biotin 342 Fab′ bindingwith an affinity of 0.09 nM (FIG. 29C). 342 Fab′ inhibits biotin 5c8binding with two affinities, 0.134 nM and 76 nM (FIG. 29C).Cross-blocking of biotin 342 by unlabeled 5c8 Fab′ is shown in FIG. 29D.The maximum concentration of 5c8 Fab′ on this curve, 50 nM, is wellabove the saturation point for 5c8 and is also well above the 1 nMconcentration of biotin 342. The lack of complete inhibition at theseconcentrations suggests that 5c8 is unable to completely block all ofthe 342 binding sites on CD40L.

1. (canceled)
 2. An anti-CD154 antibody or antigen-binding fragmentthereof, comprising: (a) a V_(H) domain sequence selected from SEQ IDNO: 1, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 and an amino acidsequence comprising one or more conservative substitutions that is atleast 90% identical to any one of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO:10 or SEQ ID NO: 11; and a V_(L) domain sequence selected from SEQ IDNO: 2 or SEQ ID NO: 14 and an amino acid sequence comprising one or moreconservative substitutions that is at least 90% identical to any one ofSEQ ID NO: 2 or SEQ ID NO: 14; or (b) a V_(H) domain sequence comprisingSEQ ID NO: 56 and a V₁ domain sequence comprising SEQ ID NO: 54; (c) aV_(H) domain sequence comprising SEQ ID NO: 60 and a V_(L) domainsequence comprising SEQ ID NO: 58; (d) a V_(H) domain sequencecomprising SEQ ID NO: 65 and a V_(L) domain sequence comprising SEQ IDNO: 62; (e) a V_(H) domain sequence comprising SEQ ID NO: 66 and a V_(L)domain sequence comprising SEQ ID NO: 63; (f) a V_(H) domain sequencecomprising SEQ ID NO: 71 and a V_(L) domain sequence comprising SEQ IDNO: 68; (g) a V_(H) domain sequence comprising SEQ ID NO: 72 and a V_(L)domain sequence comprising SEQ ID NO:
 69. 3. The anti-CD154 antibodyaccording to claim 2 comprising V_(H) and V_(L) domain sequencesselected from: (a) SEQ ID NO: 1 or an amino acid sequence comprising oneor more conservative substitutions that is at least 90% identical to SEQID NO: 1 and SEQ ID NO: 2 or an amino acid sequence comprising one ormore conservative substitutions that is at least 90% identical to SEQ IDNO: 2, respectively; (b) SEQ ID NO: 11 or an amino acid sequencecomprising one or more conservative substitutions that is at least 90%identical to SEQ ID NO: 11 and SEQ ID NO: 2 or an amino acid sequencecomprising one or more conservative substitutions that is at least 90%identical to SEQ ID NO: 2, respectively; (c) SEQ ID NO: 9 or an aminoacid sequence comprising one or more conservative substitutions that isat least 90% identical to SEQ ID NO: 9 and SEQ ID NO: 14 or an aminoacid sequence comprising one or more conservative substitutions that isat least 90% identical to SEQ ID NO: 14, respectively; (d) SEQ ID NO: 10or an amino acid sequence comprising one or more conservativesubstitutions that is at least 90% identical to SEQ ID NO: 10 and SEQ IDNO: 14 or an amino acid sequence comprising one or more conservativesubstitutions that is at least 90% identical to SEQ ID NO: 14,respectively.
 4. The anti-CD154 antibody according to claim 2, whereinthe conservative substitution is not within the CDRs.
 5. The anti-CD154antibody or antigen-binding fragment according to claim 2, which ismodified by a covalent attachment of a functional moiety.
 6. Theanti-CD154 antibody or antigen-binding fragment according to claim 5,wherein the functional moiety is poly(ethyleneglycol) or a derivativethereof.
 7. The anti-CD154 antibody or antigen-binding fragmentaccording to claim 6, wherein the antigen-binding fragment is a Fab′wherein a thiol group in a modified hinge region is covalently linked toa maleimide group that is covalently linked to a lysine residue, whereina methoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da is attached to each of the amine groups of thelysine.
 8. The anti-CD154 antibody according to claim 2, wherein theantibody comprises an immunoglobulin Fe region selected from IgG1, IgG2,IgG3 and IgG4 Fc regions, or derived from IgG1, IgG2, IgG3 or IgG4 Fcregions.
 9. The anti-CD154 antibody according to claim 2, wherein thebinding protein further comprises a variant Fc region that confersreduced effector function compared to a native or parental Fc region.10. The anti-CD154 antibody according to claim 9, wherein the variant Fcregion is a hybrid Fc region comprising sequences from more than one IgFe domain type.
 11. The anti-CD154 antibody according to claim 2,wherein the antibody is not glycosylated.
 12. The anti-CD154 antibodyaccording to claim 2, wherein the antibody or antigen binding fragmentis linked to a functional moiety.
 13. An isolated nucleic acid moleculeencoding the anti-CD154 antibody of claim
 2. 14. A vector comprising anucleic acid molecule of claim
 13. 15. A host cell comprising a vectorof claim
 14. 16. A method for producing a CD154 binding proteincomprising the steps of (a) culturing a host cell of claim 15 underconditions suitable for expression of the CD154 binding protein by thehost cell; and (b) recovering the CD154 binding protein.
 17. The methodaccording to claim 16, wherein the host cell is a prokaryotic or aeukaryotic cell.
 18. A composition comprising the anti-CD154 antibodyaccording to claim 2, or an antigen binding fragment thereof, and asuitable pharmaceutical carrier.
 19. A method for treating or preventinga human condition, disorder or disease mediated in whole or in part byCD40 signaling, or a symptom of any of the foregoing, the methodcomprising the step of administering to a subject in need thereof atherapeutically effective amount of the anti-CD154 antibody of claim 2or an antigen binding fragment thereof.
 20. The method according toclaim 19, wherein the condition, disorder or disease is an inflammatoryor autoimmune response or fibrosis.
 21. The method according to claim20, wherein the inflammatory or autoimmune response or fibrosis isselected from rheumatoid arthritis, systemic lupus erythematosis,spondyloarthritis, inflammatory bowel disease, Crohn's disease,psoriasis and multiple sclerosis.